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Ube  Tftniverstts  ot  Cbtcago 

FOUNDED  BY  JOHN  D.  ROCKEFELLER 


A  STUDY  OF  THE  CONCENTRATION 

OF  THE  ANTIBODIES  IN  THE 

BODY  FLUIDS  OF  NORMAL 

AND   IMMUNE 

ANIMALS 


A  DISSERTATION 

SUBMITTED    TO    THE    FACULTY    OF     THE    OGDEN     GRADUATE    SCHOOL 

OF    SCIENCE    IN    CANDIDACY    FOR    THE    DEGREE    OF 

DOCTOR    OF    PHILOSOPHY 

(DEPARTMENT  OF  PHYSIOLOGY) 


BY 


FRANK  C.  BECHT  and  JAMES  R.  GREER 


CHICAGO 
1910 


mniperstts  of  Gbicago 

FOUNDED  BY  JOHN  D.  ROCKEFELLER 


A  STUDY  OF  THE  CONCENTRATION 

OF  THE  ANTIBODIES  IN  THE 

BODY  FLUIDS  OF  NORMAL 

AND   IMMUNE 

ANIMALS 


A  DISSERTATION 

SUBMITTED    TO    THE    FACULTY    OF     THE    OGDEN     GRADUATE    SCHOOL 

OF    SCIENCE    IN    CANDIDACY    FOR    THE    DEGREE    OF 

DOCTOR    OF    PHILOSOPHY 

(DEPARTMENT  OF  PHYSIOLOGY) 


BY 
FRANK  C.  BECHT  and  JAMES  R.  GREER 


CHICAGO 
1910 


A  STUDY  OF  THE  CONCENTRATION  OF  THE  ANTI- 
BODIES IN  THE  BODY  FLUIDS  OF  NORMAL 
AND  IMMUNE  ANIMALS.* 

FRANK   C.    BECHT   AND   JAMES    R.    GREEK. 
(From  the  Hull  Physiological  Laboratory,  The  University  of  Chicago.) 

THE  presence  of  antibodies  of  various  kinds  in  serum  has  long 
been  known,  and  the  concentration  in  that  fluid  has  been  carefully 
studied.  The  presence  of  antibodies  in  the  various  other  body 
fluids  has  not  been  so  carefully  investigated,  nor  has  sufficient  allow- 
ance been  made  for  individual  variations  in  animals  of  the  same 
species.  While  the  authors  were  associated  with  Dr.  Carlson  in  his 
work  on  lymph  formation,  he  suggested  that  a  careful  comparison 
between  the  concentration  of  the  antibodies  in  the  various  body  fluids 
of  the  same  animal  might  be  of  considerable  importance  in  determin- 
ing the  differences  between  the  lymph  and  serum,  and  in  that  way 
throw  light  on  some  of  the  problems  of  lymph  formation,  and  possibly 
also  on  the  point  of  origin  of  antibodies.  When  the  work  was  begun, 
we  intended  to  collect  lymph  from  the  different  organs,  but  the 
practical  difficulties  encountered  in  introducing  cannulae  into  the 
delicate  lymphatics  of  such  organs  as  the  spleen  was  so  great,  that 
the  project  was  temporarily  abandoned,  and  the  work  has  been  con- 
fined to  a  comparison  between  serum,  lymph  from  the  cervical  lym- 
phatics, lymph  from  the  thoracic  duct,  pericardial  fluid,  cerebrospinal 
fluid,  and  aqueous  humor.  Thus  far  work  has  been  done  on  the 
hemolysins,  hemagglutinins,  agglutinins  for  the  typhoid  bacillus, 
the  protein  precipitins,  and  the  opsonins,  bacterial  and  erythrocytic. 
No  work  has  yet  been  undertaken  on  the  bacteriolysins.  We  have 
not  enough  data  to  enable  us  to  draw  any  broad  conclusions,  and  we 
will  content  ourselves  with  presenting  what  we  believe  to  be  the  facts 
under  the  various  conditions  studied. 

Literature. — The  first  studies  that  we  were  able  to  find  on  the  relative  concentra- 
tion of  antibodies  in  the  various  body  fluids  were  those  of  Pegano,36  who  found  the 
concentration  of  hemolysins  of  the  thoracic  lymph  in  dogs  lower  than  that  of  the  serum. 
Falloise  ll  and  later  Batelli*  confirmed  the  work  of  Pegano.  Hughes  and  Carlson-8 
working  on  normal  dogs,  horses,  and  cats  found  the  concentration  of  hemolysins  for 

*  Received  for  publication  November  9,  1909. 

127 


218508 


BECHT  AND  JAMES  R.  GREER 

rabbit  corpuscles  in  the  body  fluids  to  form  a  descending  series:  serum,  thoracic  lymph, 
neck  lymph,  pericardial  fluid,  aqueous  humor.  No  lysins  were  found  in  the  cere- 
brospinal  fluid.  Straus  and  Wolf4°  studied  the  hemolytic  power  against  rabbit  cor- 
puscles of  the  cerebrospinal  fluid,  edema  fluid,  pleural  and  pericardial  transudates, 
and  blister  fluid,  and  attempted  to  correlate  the  hemolytic  strength  with  the  protein 
content.  Marshall  and  Morgenroth2s  found  anti-complement  and  anti-amboceptor 
in  a  pathological  exudate — an  ascites  fluid.  Hedinger  js  studied  the  hemolytic  power 
of  non-inflammatory  exudates  like  those  arising  from  cirrhosis  of  the  liver  and  heart 
failure,  and  found  that  they  were  not  so  hemolytic  as  the  serum.  The  inflammatory 
exudates  arising  from  cases  of  tuberculosis  and  carcinoma  were  not  so  strongly  hemolytic 
as  non-inflammatory  exudates.  He  failed  to  find  hemolysins  in  the  fluid  from  an 
ovarian  cyst,  or  in  the  cerebrospinal  fluid  in  two  cases  of  tuberculosis.  Marshall2^ 
found  that  pleural  and  ascites  fluids  were  more  strongly  hemolytic  than  the  serum 
from  an  infant.  But  no  conclusions  can  be  drawn  from  this  comparison  in  regard  to  the 
comparative  hemolytic  power  of  serum  and  other  body  fluids  in  the  same  individual. 
He  found  a  multiplicity  of  amboceptors  and  complements  in  the  fluids  that  he  studied. 
Grollo  4  could  find  no  amboceptors  for  rabbit  corpuscles  in  transudates,  but  found 
them  in  exudates,  altho  in  the  latter  complement  is  often  lacking.  He  suggests  this 
method  as  a  means  of  diagnosis  between  transudates  and  exudates.  Liidke22  con- 
firmed the  findings  of  Marshall  in  regard  to  the  hemolytic  strength  of  transudates  and 
exudates.  Granstronvs  found  wide  variations  in  the  hemolytic  content  of  transu- 
dates and  exudates,  and  could  establish  no  characteristics  essential  for  either.  The 
hemolysins  did  not  run  parallel  with  those  of  the  blood.  Isolysins  are  found  less 
frequently  in  transudates  and  exudates  than  in  the  blood.  Hemolysins  were  not 
found  in  the  cerebrospinal  fluid.  Isolysins  and  heterolysins  were  found  independent 
of  the  albumen  content,  number  of  the  leukocytes,  and  the  osmotic  pressure  of  the 
fluids  tested.  Tedeschi4;  found  precipitins  in  both  transudates  and  exudates,  less 
frequently  in  the  latter  than  in  the  former.  Mioni3°  found  amboceptor  but  no  com- 
plement for  guinea-pig  corpuscles  in  the  pericardial  fluid  of  the  ox.  Bard2  claims  to 
have  found  hemolysins  in  the  cerebrospinal  fluid  of  patients,  and  found  that  they  were 
increased  during  various  diseases.  Massaglia26  could  not  confirm  the  work  of  Bard. 
His  results  in  both  healthy  and  diseased  individuals  were  negative.  The  presence  of 
antibodies  for  syphilitic  material  in  the  cerebrospinal  fluid  has  been  shown  by  various 
investigators,  among  them  Morgenroth  and  Stertz,3i  and  Wassermann  and  Plaut.42 
Gatti  2  could  demonstrate  no  hemolysins  in  the  aqueous  humor  of  the  ox.  Levaditi 2 
showed  that  there  is  normally  no  opsonin  in  the  aqueous  humor;  but  if  the  fluid  of  the 
anterior  chamber  of  the  eye  of  an  immune  animal  is  withdrawn,  the  newly  formed 
aqueous  humor  will  contain  opsonin.  Bohme6  investigated  the  opsonin  content  of 
pleural,  peritoneal,  and  abscess  fluids  He  found  that  usually  in  such  cases  the  opsonin 
content  of  the  fluid  was  reduced  for  the  infecting  organism,  but  remained  unchanged 
for  other  bacteria.  He  could  find  no  opsonin  in  normal  cerebrospinal  fluid,  but  found 
them  there  after  an  inflammation  had  been  set  up  in  the  dura.  He  could  not  develop 
opsonins  in  the  cerebrospinal  fluid  by  repeated  puncture  as  Levaditi  had  done  by 
drawing  off  the  aqueous  humor.  He  believes  that  there  is  a  relation  between  the 
protein  content  and  the  opsonin  action  of  a  fluid. 

Methods. — The  plan  of  study  adopted  was  to  determine  first  the  concentration 
of  the  antibodies  in  the  body  fluids  of  normal  cats  and  dogs;  then  the  concentration  in 
actively  immunized  animals;  and,  finally,  to  study  the  passage  of  the  antibodies  from 


CONCENTRATION  OF  ANTIBODIES  129 

the  blood  into  the  other  body  fluids  in  animals  passively  immunized  by  the  with- 
drawal of  large  quantities  of  blood,  and  the  injection  of  a  corresponding  amount  of 
warm,  defibrinated  blood  from  an  actively  immunized  animal. 

The  body  fluids  were  secured  under  as  nearly  aseptic  conditions  as  possible.  The 
animal  was  anesthetized  with  ether,  and  kept  in  a  state  of  complete  anesthesia,  by  the 
administration  of  the  vapor  through  a  trachea  cannula  or  through  a  tube  introduced 
through  the  larynx.  The  neck  lymphatics  were  then  isolated,  and  small,  sterile,  glass 
cannulae  provided  with  sterile,  rubber  tubing  were  inserted.  If  there  was  no  free  flow 
of  lymph,  the  neck  was  gently  massaged.  The  lymph  was  never  allowed  to  come  in 
contact  with  the  air  of  the  room,  for  as  soon  as  it  filled  the  cannula  and  a  part  of  the 
rubber  tubing,  it  was  drawn  off  by  means  of  a  fine,  sterile  Pasteur  pipette,  and  placed 
in  a  dry,  sterile  test  tube  plugged  as  for  bacteriological  work.  The  lymph  was  allowed 
to  coagulate  spontaneously  in  the  test  tube,  and  was  then  defibrinated,  and  the  delicate 
coagulum  removed. 

The  thoracic  duct  was  tied  off  at  the  same  time  as  the  isolation  of  the  neck  lym- 
phatics so  that  the  lymph  formed  during  the  experiment  was  retained  in  the  duct.  Usually 
the  lymph  from  this  duct  was  not  collected  until  the  animal  had  been  bled  to  death, 
altho  sometimes  it  was  collected  simultaneously  with  the  neck  lymph.  The  routine 
method  was  to  draw  the  lymph  by  means  of  a  Pasteur  pipette  provided  with  a  bulb,  so 
that  the  fluid  never  came  in  contact  with  the  air  at  all.  This  fluid  was  also  defibrinated. 

The  pericardial  fluid  was  never  collected  until  after  the  death  of  the  animal  by  very 
complete  bleeding  from  the  arteries  and  veins  of  the  neck.  The  thorax  was  opened 
by  removing  the  sternum,  a  small  hole  was  cut  into  the  pericardium,  and  the  fluid  was 
removed  by  means  of  a  sterile  Pasteur  pipette. 

We  found  it  a  good  plan  in  our  experiments  to  suspend  the  animal  by  the  jaws  for 
a  few  minutes  before  attempting  to  withdraw  the  cerebrospinal  fluid.  This  drained 
away  the  blood  from  the  head  and  made  admixture  of  this  fluid  with  blood  less  likely. 
Our  method  was  to  open  the  dura  between  the  first  and  the  second  cervical  vertebrae, 
and  then  remove  the  fluid  by  means  of  the  Pasteur  pipette.  The  end  of  the  pipette 
must  be  well  rounded  in  the  flame,  otherwise  rupture  of  the  delicate  blood  vessels  of 
the  meninges  is  likely  to  follow. 

The  method  of  collecting  the  aqueous  humor  was  simple  and  easy.  It  consisted 
in  thrusting  a  sharp  pointed  Pasteur  pipette  into  the  anterior  chamber  of  the  eye 
through  the  cornea,  and  allowing  the  aqueous  humor  to  flow  into  it,  largely  by  the 
tension  within  the  eyeball.  A  little  suction  sufficed  to  remove  the  last  drop  of  the  fluid. 

The  serum  was  secured  from  blood  drawn  when  the  animal  was  bled  to  death, 
and  in  most  cases  was  freed  from  the  corpuscles  at  once. 

Careful  notes  were  made  in  regard  to  the  condition  of  the  fluids,  and  in  most 
cases  where  there  was  any  admixture  of  blood,  the  fluid  was  discarded. 

HEMOLYSIS   AND   HEMAGGLUTININS. 

Methods. — The  study  of  the  hemolysins  and  hemagglutinins  in  normal*  animals 
was  made  on  dogs  only.  The  animals  utilized  were  brought  in  from  various  parts  of 
the  city.  Most  of  the  tests  were  made  with  rabbit  corpuscles  in  5  per  cent  suspension 
in  0.9  per  cent  NaCl  solution.  In  some  cases  rat  and  horse  corpuscles  were  used. 

*The  term  "normal"  means  animals  which  had  not  previously  been  immunized  by  us.  We  had 
no  way  of  knowing  what  their  history  had  been  previous  to  coming  to  the  laboratory. 


130  FRANK  C.  BECHT  AND  JAMES  R.  GREEK 

Our  methods  were  the  following:  Quantities  of  the  various  body  fluids  of  the  animal 
to  be  tested  varying  between  o.  i  c.c.  and  o.oooi  c.c.  were  placed  in  a  series  of  eight 
dry,  sterile  test  tubes  plugged  as  for  bacteriological  work.  In  order  to  make  the 
necessary  measurements  with  a  pipette  graded  to  Tfa  of  a  c.c.,  dilutions  of  the  body 
fluids  TV  and  ^  were  made.  To  the  fluid  in  each  tube  enough  sterile  0.9  per  cent 
NaCl  solution  was  added  to  make  the  total  volume  up  to  0.4  c.c.  To  this  was  then 
added  o.  2  c.c.  of  a  5  per  cent  suspension  of  the  corpuscles  to  be  tested.  In  this  way  we 
got  dilutions  of  the  fluid  varying  between  i :  6  and  i :  6, 144.  All  of  the  fluids  from  the 
same  animal  were  prepared,  the  tubes  were  placed  in  a  block  containing  a  suitable 
number  of  holes,  and  adjusted  to  the  sliding  platform  of  a  shaker  in  an  incubator 
warmed  to  37°  C.  The  shaker  was  run  by  water  power,  and  the  motion  was  rapid 
enough  to  secure  constant,  thorough  agitation,  but  not  violent  enough  to  injure  the 
corpuscles.  The  routine  technic  was  to  keep  the  tubes  in  the  shaker  for  an  hour  and 
in  the  ice-box  from  12  to  20  hours  to  permit  sedimentation  of  the  corpuscles  before  the 
final  reading. 

In  determining  the  amount  of  hemolysis  in  the  final  reading,  the  following  method 
was  employed:  A  measured  sample  of  the  corpuscle  suspension  in  the  test  was  sedi- 
mented  in  the  centrifuge,  and  the  supernatant  liquid  drawn  off  with  a  pipette.  The 
corpuscles  were  then  laked  by  adding  distilled  water  to  restore  the  original  volume. 
This  sample  contained  three  times  as  much  hemoglobin  as  the  hemolytic  tests,  because 
0.2  c.c.  of  the  corpuscles  were  added  to  0.4  c.c.  of  the  fluid  tested.  Therefore,  the 
above  sample  was  diluted  to  three  times  its  volume  with  water.  This,  then,  would  give 
exactly  the  same  concentration  of  hemoglobin  as  in  any  tube  in  the  test,  provided  that 
the  hemolysis  was  complete,  and  is  termed  100  per  cent  for  this  sample  of  corpuscles. 
By  further  dilution  tubes  containing  90  per  cent,  80  per  cent,  etc.,  were  prepared. 
No  attempt  was  made  to  estimate  closer  than  10  per  cent.  A  new  scale  was  made  for 
each  sample  of  corpuscles. 

The  agglutinins  were  read  from  the  same  tubes  as  the  hemolysins.  The  method 
employed  to  determine  whether  or  not  agglutination  had  occurred,  was  inspection  of 
the  rim  of  sedimented  corpuscles.  When  the  corpuscles,  after  sedimentation  by  stand- 
ing in  the  ice-box,  show  a  perfectly  smooth,  knife-edged  border,  no  agglutination  has 
occurred.  If  the  border  is  slightly  or  decidedly  roughened,  agglutination  has  occurred 
At  first  this  method  was  carefully  supplemented  by  microscopic  examination,  but  it 
was  soon  found  so  accurate  that  in  the  later  experiments  we  depended  entirely  upon 
the  observation  of  the  rim  of  the  corpuscles,  and  dispensed  with  the  use  of  the  micro- 
scope. 

A.  Normal  animals. — The  concentration  of  lysins  and  hemag- 
glutinins  in  the  body  fluids  of  normal  animals  varies  within  rather 
narrow  limits.  This  variation  is  great  enough,  however,  to  make  it 
necessary  that  the  comparison  be  made  between  the  body  fluids  of 
the  same  animal.  The  following  experiment  shows  the  behavior 
of  the  body  fluids  of  the  normal  dog. 

Table  I  shows  that  the  concentration  of  hemolysins  is  greater 
in  the  serum  than  in  the  other  body  fluids;  thoracic  lymph  is  next, 
at  least  in  the  case  of  rabbit  corpuscles;  and  neck  lymph  is  third. 


CONCENTRATION  OF  ANTIBODIES 


This  difference  in  the  hemolytic  power  of  serum  or  other  body  fluid 
against  the  corpuscles  of  different  species  of  animals  has  been 
explained  by  Ehrlich  and  his  coworkers  on  the  basis  of  a  multiplicity 
of  amboceptors  and  complements,  some  of  which  are  specific,  some 

TABLE  i. 

COMPARATIVE  HEMOLYTIC  AND  AGGLUTINATING  POWER  OF  THE  BODY  FLUIDS  OF  A  NORMAL  DOG  ON 
RABBIT  AND  RAT  CORPUSCLES. 


DILUTION 

SERUM 

NECK  LYMPH 

THORACIC  LYMPH 

Rabbit 

Rat 

Rabbit 

Rat 

Rabbit 

Rat 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:6  

IOO 

40 
o 

0 
0 
0 

+ 
+ 

0 
0 

o 

10 

0 

o 

0 

sp* 

0 

+ 

0 
0 
0 

0 

5 

0 
0 
0 
0 
0 

+ 

+ 

0 
0 
0 

o 

0 
0 
0 
0 

sp 
o 

0 
0 
0 
0 

o 

40 

0 
0 
0 
0 

o 

+ 
+ 
+ 
+ 

0 

o 

0 
0 
0 

sp 

0 

o 

+ 
+ 

+ 

0 
0 

:24  
=  48 

:g6  
:384  

DILUTION 

PERICARDIAL  FLUID 

CEREBROSPINAL  FLUID 

AQUEOUS  HUMOR 

Rabbit 

Rat 

Rabbit 

Rat 

Rabbit 

Rat 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:6  
:i2  
'.24  
:48  
•96  
:3§4  

0 

o 

0 
0 
0 
0 

+ 
+ 

0 

o 

0 
0 

0 
0 

sp 
o 

0 

o 

0 
0 

o 

0 

o 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 

0 

sp 

0 

o 
sp 
o 

0 

0 
0 

o 

0 
0 
0 
0 
0 

o 

0 
0 
0 
0 
0 

o 

sp 
sp 

0 

sp 

0 

o 

o 

0 
0 

*It  will  be  noted  that  in  this  table  several  tubes  are  marked  "sp."  By  that  symbol  is  meant 
hemolysis  not  due  to  the  ordinary  hemolysins.  The  appearance  of  the  partially  laked  corpuscles  is  entirely 
different  from  that  in  the  ordinary  hemolytic  test.  The  hemoglobin  can  be  seen  diffusing  from  the  sedi- 
mented  corpuscles,  while  the  supernatant  fluid  remains  perfectly  clear.  The  hemoglobin  has  the  peculiar 
reddish  purple  tint  of  reduced  hemoglobin,  instead  of  the  clear  red  of  oxyhemoglobin.  Furthermore, 
laking  may  appear  anywhere  in  the  series,  frequently,  where  no  hemolysis  is  to  be  expected,  and  is  met 
more  often  in  fluids  like  the  cerebrospinal,  or  aqueous  humor,  which  are  normally  not  hemolytic,  than  in 
the  other  fluids.  Rat  corpuscles  seem  more  susceptible  to  this  form  of  hemolysis  than  rabbit  corpuscles. 
Complement  seems  to  inhibit  this  form  of  hemolysis. 

non-specific.  In  most  cases  the  thoracic  lymph  of  normal  dogs  is 
hemolytic  for  rat  corpuscles,  altho  in  the  experiment  cited  above,  such 
was  not  the  case. 

As  may  be  seen  from  the  table  above,  the  concentration  of  agglu- 
tinins  may  be  higher  in  the  thoracic  lymph  than  in  the  serum.  Such, 
however,  is  not  the  usual  finding.  In  10  experiments  on  normal 
dogs  we  found  in  seven  the  concentration  of  agglutinins  highest  in 
the  serum;  in  two  it  was  highest  in  the  thoracic  lymph;  and  in  one 


132  FRANK  C.  BECHT  AND  JAMES  R.  GREER 

the  concentration  was  the  same  in  both.  The  fact  that  the  concen- 
tration of  agglutinins  may  be  greater  in  the  thoracic  lymph  than  in 
the  serum,  renders  it  hard  to  see  how  these  antibodies  can  come  from 
the  blood  by  pure  nitration,  for  in  that  case,  we  should  expect  the 
hemolysins  to  run  a  parallel  course — a  thing  which  they  do  not  do — 
or  else  we  must  assume  that  the  agglutinins  pass  through  membranes 
more  readily  than  the  hemolysins.  It  would  be  necessary,  also,  on 
the  basis  of  filtration,  to  assume  sudden  great  changes  in  the  concen- 
tration of  the  agglutinins  in  the  blood,  for  on  no  other  basis  could  we 
explain  the  fact  that  the  concentration  of  agglutinins  would  be  so 
much  lower  in  the  serum  by  the  time  the  lymph  reached  the  upper 
end  of  the  thoracic  duct,  than  it  was  at  the  time  the  lymph  was 
formed.  Of  course  other  explanations  are  possible:  there  may  be 
an  active  secretion  of  the  agglutinins  into  the  lymph  from  the  blood, 
or  the  agglutinins,  after  being  formed  in  the  area  drained  by  the 
thoracic  duct,  are  thrown  into  the  lymph,  reaching  the  blood  by  that 
route.  Much  more  investigation  must  be  made  before  any  conclusion 
can  be  reached  on  this  point. 

The  pericardial  fluid  when  collected  under  the  best  conditions 
never  shows  hemolysins  for  rabbit  corpuscles.  Agglutinins  may  or 
may  not  be  present.  In  four  of  our  ten  supposedly  normal  dogs 
hemolysis  was  noted,  in  only  one  case  amounting  to  more  than  10  per 
cent.  Of  these  four  animals,  two  were  in  poor  condition,  emaciated, 
and  generally  run  down,  and  both  these  dogs  yielded  excessive 
amounts  of  pericardial  fluid;  in  the  other  two  cases,  the  pericardial 
was  found  to  contain  a  few  erythrocytes.  Agglutinins  were  found 
in  all  four  of  these  cases  and  in  three  others,  making  a  total  of 
seven  in  ten.  From  these  experiments  we  are  inclined  to  believe  that 
hemolysins  are  not  found  in  the  pericardial  fluid  of  normal  dogs.  The 
fact  that  some  animals  showed  hemolysins  in  the  pericardial  fluid 
we  would  explain  as  a  pericardial  transudate  in  two  cases,  and  to 
admixture  with  blood  in  two  cases.  We  did  not  test  whether  it  was 
amboceptor,  or  complement,  or  both  which  was  absent  from  the 
fluid,  altho  we  have  evidence  on  this  point  in  immune  animals. 
Agglutinins  for  rabbit  corpuscles  may  or  may  not  be  present  in  the 
pericardial  fluid  of  normal  dogs. 

As  will  be  seen  from  Table  i  the  cerebrospinal  fluid  and  aqueous 


<x  r 


CONCENTRATION  or  ANTIBO^ES^  133 


humor  of  normal  animals  contain  no  lysins  or  agglutinins  for  rat  or 
rabbit  corpuscles.  In  our  10  experiments  on  normal  animals  there 
were  no  traces  of  hemolysis  or  agglutination  in  a  single  case  where 
admixture  of  blood  was  eliminated.  Our  results  with  cerebrospinal 
fluid  confirm  those  of  Massaglia,  who  could  find  no  lysins  in  that 
fluid,  and  are  contrary  to  those  of  Bard,  who  claims  to  have  demon- 
strated them  there. 

Conclusions.  —  i.  In  the  normal  dog  hemolysins  for  rabbit  cor- 
puscles are  found  in  the  serum,  neck  lymph,  and  thoracic  lymph, 
but  are  absent  from  the  pericardial  fluid,  cerebrospinal  fluid,  and 
aqueous  humor.  They  are  most  concentrated  in  the  serum,  less 
concentrated  in  the  thoracic  lymph,  and  are  found  only  in  traces  in 
the  neck  lymph. 

2.  Agglutinins  are  found  in  the  serum,  neck  lymph,  and  thoracic 
lymph  of  normal  dogs.     They  may  or  may  not  be  present  in  the 
pericardial  fluid,  and  are  always  absent  from  cerebrospinal  fluid 
and  aqueous  humor.     In  most  cases  the  concentration  descends  in 
the  following  order:  serum,  thoracic  lymph,  neck  lymph,  pericardial 
fluid;  altho  in  some  cases,  the  order  is  thoracic  lymph,  serum,  neck 
lymph,  pericardial  fluid. 

3.  Serum  and   thoracic  lymph  show  a  weaker  hemolysis  and 
agglutination    toward    rat    than    toward    rabbit    corpuscles.     Neck 
lymph  lakes  and  agglutinates  rabbit  but  not  rat  corpuscles.     Peri- 
cardial fluid  agglutinates  rabbit  but  not  rat  corpuscles.     Cerebro- 
spinal fluid  and  aqueous  humor  neither  lake  nor  agglutinate  rat  or 
rabbit  corpuscles. 

B.  Immunized  animals.  —  Various  methods  of  producing  active 
immunity  were  employed  with  good  success.  It  is  of  interest  to 
ascertain  what  methods  of  immunizing  yield  the  best  results.  We 
employed  the  following:  (i)  Immunization  of  dogs  with  rabbit  blood  : 
(a)  intraperitoneally  by  a  single  large  injection  of  from  80  to  150  c.c. 
of  blood,  (b)  intraperitoneally  by  repeated  small  injections,  (c)  sub- 
cutaneously  by  repeated  small  injections.  (2)  Immunization  of  dogs 
with  horse  serum:*  (a)  by  a  single  large  intraperitoneal  injection  of 
100-150  c.c.,  (b)  by  repeated,  small  intraperitoneal  injections,  (c)  by 

*  This  serum  was  secured  aseptically,  November,  1908,  by  drawing  the  blood  from  the  carotid  of 
a  horse  into  jars.  It  was  allowed  to  coagulate  and  stand  in  the  ice-box  until  the  serum  came  out.  The 
serum  was  then  sealed  into  bulbs  and  kept  in  the  ice-box  until  used. 


134 


FRANK  C.  BECHT  AND  JAMES  R.  GREER 


repeated,  small  subcutaneous  injections.  A  comparison  of  the 
results  secured  by  using  the  fluids  directly  from  the  animal,  so  far  as 
the  lysins  are  concerned,  is  not  conclusive,  for,  as  will  be  seen,  com- 
plement is  not  increased,  at  least  not  in  proportion  to  the  ambocep- 
tors,  if  at  all.  This  fact  necessitates  the  use  of  sufficient  complement 
to  supply  all  the  amboceptors  present  to  demonstrate  the  true  state 
of  affairs.  So  far  as  the  agglutinins  are  concerned,  apparently  a 
single  large  dose  of  the  serum  or  blood  may  develop  them  more 
markedly  than  the  other  methods  tried.  The  repeated,  small  intra- 
peritoneal  injections  yielded  the  most  uniform  results. 

As  has  been  noted  by  numerous  investigators,  the  increase  in 
complement  does  not  keep  pace  with  the  increase  in  amboceptors. 
The  apparent  increase  in  the  hemolysins  and  in  the  agglutinins  for 
rabbit  corpuscles  is  shown  in  Table  2. 

TABLE  2. 

LYTIC  AND  AGGLUTINATING  ACTION  OF  THE  BODY  FLUIDS  OF  A  DOG  IMMUNIZED  WITH  RABBIT  BLOOD. 

(November  25,  December  15,  10  c.c.  rabbit  blood  intraperitoneally;  December  15, 

15  c.c.     Fluids  collected  December  23.) 


DILUTION 

SERUM 

NECK 
LYMPH 

THOKACIC 

LYMPH 

PERICAR- 
DIAL  FLUID 

CEREBRO- 

SPINAL 

FLUID 

AQUEOUS 
HUMOR 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:i*  
'•3  
•6 

100 
100 

50 
o 

0 

o 

0 

o 

+ 
+ 
+ 
+ 
+ 
+ 
+ 

0 

60 

0 

o 

0 

o 

0 

o 

0 

+ 
+ 
+ 
+ 
+ 
+ 
o 

0 

IOO 

60 
o 

0 

o 

0 

o 

0 

+ 
+ 
+ 
+ 
+ 
+ 

0 
0 

0 
0 
0 
0 

+ 
+ 
+ 
+ 

0 
0 
0 
0 

o 

0 

o 

0 

0 
0 
0 
0 

o 

0 

o 

0 

:i2  
=  24  
:48  
:p6  
:3»4  
'.1,536  
:6,i44  

From  Table  2  it  can  be  seen  by  comparison  with  Table  i  that  the 
repeated  injections  of  rabbit  blood  does  not  appear  to  increase  to  any 
very  marked  extent  the  hemolytic  power  of  the  body  fluids  over  that 
of  a  normal  animal. 

There  is,  however,  a  marked  increase  in  the  power  of  the  body 
fluids  to  agglutinate  rabbit  corpuscles  in  those  fluids  which  had  the 
power  to  agglutinate  them  previous  to  the  injection;  there  is  no 
development,  except  in  a  few  cases,  of  agglutinins  in  the  cerebrospinal 
fluid  and  the  aqueous  humor.  Altho  Table  2  does  not  show  this 
point,  a  careful  comparison  of  this  table  with  the  succeeding  ones 


CONCENTRATION  OF  ANTIBODIES 


will  show  that  the  relative  concentration  of  the  agglutinins,  in  the 
body  fluids,  remain  the  same  during  the  process  of  immunization. 

That  there  is,  however,  a  marked  increase  in  the  amboceptor 
content  of  the  body  fluids,  normally  containing  them,  during  the 
process  of  immunization  is  shown  by  Table  3. 

TABLE  3. 

LYTIC  AND  AGGLUTINATING  POWER  ON  RABBIT  CORPUSCLES  OF  THE  BODY  FLUIDS  OF  DOG  IMMUNIZED 

WITH  RABBIT  BLOOD  AS  AFFECTED  BY  COMPLEMENT  (0.2  c.c.  FRESH  GUINEA-PIG  SERUM). 

(Intraperitoneal  injections  of  rabbit  blood  as  follows:  December  i,  5  c.c.;  December  5,  7^  c.c.; 

December  10,  10  c.c.;  December  15,  15  c.c.;  December  21,  16  c.c.;  January  16,  20  c.c. 

Fluids  collected  February  8.) 


DILUTION 

SERUM 

NECK  LYMPH 

THORACIC  LYMPH 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:6  

100 
100 

90 

10 
0 

o 

0 

o 

+ 
+ 
+ 
+ 

0 

o 

100 
100 
100 
100 

So 
30 

10 

o 

+ 
+ 

0 

o 

60 

10 
0 
0 
0 
0 
0 
0 

+ 
+ 
+ 
4- 

0 
0 
0 

o 

E 

40 

20 
0 
0 
0 

o 

+ 
+ 
+ 
+ 

0 
0 
0 
0 

100 

7o 
20 

0 

o 

0 
0 
0 

+ 
+ 

+ 

0 

o 

0 

oooooooo 

+ 
+ 

+ 

0 
0 

o 

:24  
=  48 

:g6  

:384 

:  1,336........ 

DILUTION 

PERICARDIAL  FLUID 

CEREBROSPTNAL  FLUID 

AQUEOUS  HUMOR 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:6  
:i2  
=  24  
=  48  
:96  

0 

o 

0 
0 
0 

+ 
+ 
+ 

+ 

0 

20 
10 
0 
O 
0 

+ 
+ 
+ 
+ 

0 

0 
0 
0 
0 

0 
0 
0 
0 

o 

0 
0 
0 

0 
0 
0 
0 

0 
0 
0 
0 

0 
0 
0 
0 

0 

o 

0 
0 

o 

0 
0 

o 

1384  
:  1,536  

:6,i44  

This  experiment  shows  very  clearly  that  in  the  immunized  animal 
the  serum,  neck  lymph,  thoracic  lymph,  and  pericardial  fluid  do  not 
contain  complement  in  sufficient  quantity  to  activate  all  of  the  ambo- 
ceptor present  in  the  fluid,  because  the  addition  of  guinea-pig  com- 
plement,* in  doses  of  itself  not  ly tic,  is  able  to  produce  stronger 

*  We  experienced  considerable  difficulty  in  securing  a  complement  which  was  effective  and  at  the 
same  time  did  not  of  itself  produce  hemolysis.  Rabbit  serum,  guinea-pig  serum,  and  dog  serum  from 
which  the  amboceptor  had  been  separated  in  the  cold  were  tried.  The  guinea-pig  serum  proved  the  only 
effective  one. 


136      FRANK  C.  BECHT  AND  JAMES  R.  GREER 

hemolysis  where  only  traces  had  appeared,  and  to  produce  hemolysis 
in. other  cases  where  there  were  no  traces  with  exactly  the  same 
amount  of  the  fluid  without  complement. 

The  addition  of  c6mplement  in  a  non-hemolytic  dose  is  able  to 
cause  hemolysis  in  the  pericardial  fluid.  In  8  of  13  experiments  on 
blood-immune  dogs  traces  of  hemolysis  occurred  in  the  lowest  dilu- 
tions of  that  fluid.  In  these  experiments  we  could  detect  no  contami- 
nation with  blood,  neither  did  we  note  that  the  fluid  was  present  in 
excessive  amounts.  Apparently,  then,  in  dogs  immune  to  a  foreign 
blood,  amboceptor  is  always,  and  complement  usually  present  in  the 
pericardial  fluid.  This  agrees  in  part  with  the  findings  of  Mioni 
who  found  amboceptor  but  no  complement  for  guinea-pig  corpuscles 
in  the  pericardial  fluid  of  the  ox.  No  amboceptors  are  found  in  the 
cerebrospinal  fluid  or  aqueous  humor,  for  no  hemolysis  occurs  when 
effective  complement  is  added,  at  least  a  complement  which  proved 
effective  in  the  case  of  the  other  body  fluids. 

The  agglutinins  in  the  various  body  fluids  are  seen  from  Table  5 
to  run  practically  the  same  course  as  in  the  normal  animal,  except 
that  the^concentration  is  highest  in  the  serum,  a  little  lower  in  the 
thoracic  lymph,  still  lower  in  the  neck  lymph,  and  lowest,  but  always 
present,  in  the  pericardial  fluid.  Sometimes,  as  in  the  normal  animal, 
the  concentration  of  agglutinins  in  the  thoracic  lymph  is  equal  to  or 
greater  than  that  of  the  serum.  In  the  16  immune  animals  in  which 
we  compared  serum  and  thoracic  lymph,  in  n  cases  the  serum  was 
higher  in  the  concentration  of  the  agglutinins  than  the  thoracic 
lymph,  in  two  cases  the  thoracic  lymph  was  higher  than  the  serum, 
and  in  three  cases  the  two  fluids  showed  equal  concentration.  The 
fact  that  the  thoracic  lymph  may  contain  these  antibodies  in  equal  or 
even  considerably  higher  concentration  than  the  corresponding  serum 
may  be  of  significance  as  bearing  upon  the  source  of  these  substances. 

In  15  experiments  with  cerebrospinal  fluid  from  immune  dogs, 
a  positive  agglutination  was  secured  in  two.  In  16  experiments  with 
the  aqueous  humor,  positive  results  were  secured  in  five.  Thus  it 
will  be  seen  that  agglutinins  may  be  found  in  the  cerebrospinal  fluid 
and  the  aqueous  humor,  but  their  presence  is  the  exception  and  not 
the  rule. 

A  point  of  considerable  interest  is  the  determination  whether  the 


CONCENTRATION  OF  ANTIBODIES 


fluids  of  an  animal  immune  to  one  kind  of  blood  show  an  increased 
hemolytic  and  agglutinating  power  toward  the  corpuscles  of  another 
species.  An  experiment  of  this  kind  is  shown  in  Table  4. 

TABLE  4. 

LYTIC  AND  AGGLUTINATING  ACTION  ON  HORSE  AND  RABBIT  CORPUSCLES  OF  BODY  FLUIDS  OF  A  DOG 

INJECTED  WITH  HORSE  SERUM. 
(100  c.c.  of  serum  injected  intraperitoneally  January  16;  fluids  removed  January  29.) 


DILUTION 

SERUM 

NECK  LYMPH 

THORACIC  LYMPH 

Horse 
Corpuscles 

Rabbit 
Corpuscles 

Horse 
Corpuscles 

Rabbit 
Corpuscles 

Horse 
Corpuscles 

Rabbit 
Corpuscles 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

•6 

100 
100 
100 

70 
30 

0 
0 

o 

+ 
+ 
+ 
+ 
+ 

100 
100 

30 

IO 
0 

o 

0 
0 

+ 
+ 

4- 

0 
0 
0 

100 

90 
60 

IO 
0 

o 

0 
0 

4- 
4- 
+ 

+ 
4- 

0 
0 

So 

IO 
0 
0 
0 
0 
0 
0 

+ 
+ 
+ 

0 
0 
0 

o 

0 

100 
100 

5° 

20 

o 

0 

o 

0 

1  1  4-44  +  +  o 

100 

40 
o 

0 

o 

0 

o 

0 

+ 
+ 

0 

o 

0 

o 

0 

:i2  

124 

•J'.'.  
•96  
:384  

:i,536  
:6,i44  

DILUTION 

PERICARDIAL  FLUID 

CEREBROSPINAL  FLUID 

AQUEOUS  HUMOR 

Horse 
Corpuscles 

Rabbit 
Corpuscles 

Horse 
Corpuscles 

Rabbit 
Corpuscles 

Horse 
Corpuscles 

Rabbit 
Corpuscles 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:6  

•48.".!'.".;".". 

=  96  
;3846-  

80 
30 
o 

0 

o 

0 

+ 
+ 
+ 
+ 
+ 

0 

o 

0 

o 

0 

o 

0 

4- 
0 
0 
0 

o 

0 

0 
0 

o 
sp 

0 

o 

0 

o 
o 

o 

0 

o 

0 
0 

o 

0 

o 

0 
0 

o 

0 

o 

0 

o 

o 

0 
0 
0 
0 

0 
0 

o 

0 

o 

0 
0 

o 

0 

o 

Horse  corpuscle  control =o.     Rabbit  corpuscle  control =o. 

The  animal  tested  here,  while  showing  a  marked  immunity  to 
horse  corpuscles,  does  not  show  immunity  toward  rabbit  corpuscles 
much  higher  than  the  normal.  Indeed,  in  the  10  normal  animals 
studied,  in  two  cases  the  lysins  in  the  neck  lymph  were  as  concentrated 
as  here;  and  in  five  cases  the  lysins  in  the  thoracic  lymph  were  as 
concentrated  as  here.  Therefore,  the  lysins  for  rabbit  corpuscles 
are  apparently  little  more  concentrated  as  may  be  seen  by  comparing 
Table  4  with  Table  i.  The  agglutinins  are  somewhat  higher  than 
normal.  Apparently  the  immunity  is  not  entirely  specific. 

Muir  and  Browning32  have  advanced  some  evidence  that  a  com- 


138 


FRANK  C.  BECHT  AND  JAMES  R.  GREER 


plement-like  body  plays  a  role  in  agglutination.  They  used  ox 
corpuscles,  rabbit  serum  immune  to  ox  blood,  and  guinea-pig  serum 
as  complement.  They  do  not  make  the  claim  that  the  complement 
and  the  agglutinin  are  identical,  merely  that  this  form  of  complement 
behaves  like  hemolytic  complement,  is  thermostable,  and  acts  only 
when  suitable  amboceptor  is  present.  They  are  not  sure  whether  or 
not  this  is  the  same  complement  concerned  in  hemolysis.  In  the 
course  of  our  experiments  we  found  some  evidence  which  points  in  the 
opposite  direction;  viz.,  addition  of  complement  or  at  least  of  rabbit 
serum,  as  in  one  experiment,  inhibits  the  agglutination  of  rabbit 
corpuscles  by  the  fluids  of  an  immune  dog. 

TABLE  5. 
THE  INHIBITION  OF  THE  AGGLUTINATION  OF  RABBIT  CORPUSCLES  BY  THE  BODY  FLUIDS  OF  AN  IMMUNE 

DOG  BY  THE  ADDITION  OF  RABBIT  SERUM. 

(150  c.c.  rabbit  blood  intraperitoneally,  February  16.     Fluids  drawn  February  26. 
o.i  c.c.  rabbit  serum  as  complement.) 


DILUTION 

SERUM 

NECK  LYMPH 

THORACIC  LYMPH 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:6  

100 
100 

80 

10 
0 
0 
0 
0 

+ 
+ 
+ 
+ 
+ 
+ 

100 
IOO 
100 

70 

10 

o 

0 

o 

+ 
+ 
+ 

0 

o 

IOO 
10 
0 

o 

0 
0 
0 
0 

+ 

+ 
+ 
+ 
+ 
+ 

0 

IOO 

30 

0 
0 
0 
0 
0 
0 

000  +  +  +  +  1 

IOO 
IOO 

40 

0 

o 

0 
0 
0 

+ 
+ 
+ 
+ 
+ 
+ 

IOO 
IOO 
IOO 
10 
0 
0 
0 
0 

o  o  +  +  +  |  |  | 

:i2  
124  

=  48  
:96  

:384  
11,536  

:  6,144  

DILUTION 

PERICARDIAL  FLUID 

CEREBROSPINAL  FLUID 

AQUEOUS  HUMOR 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

No  Comple- 
ment 

Complement 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

Lysis 

Aggl. 

:6  
:i2  
:24 

20 
0 
0 

sp 

0 

sp 

+ 
+ 
+ 

+ 

20 
10 
0 

o 

0 

o 

+ 
+ 
+ 
+ 

0 
0 

o 

0 

o 

0 

+ 

+ 
+ 
+ 

o 

0 

o 

0 

0 
0 

o 

0 

sp 

0 

o 

0 

0 

o 

0 

0 

o 

0 
0 

0 

o 

0 

o 

:4s  :..: 

:96  

'384  
:  i,536  
:  6,144  

Corpuscle  control=o.     Complement  control=o. 

This  experiment  shows  that  the  addition  of  rabbit  serum  as  com- 
plement instead  of  favoring  agglutination  as  in  the  case;  observed  by 


CONCENTRATION  OF  ANTIBODIES  139 

Muir  and  Browning  actually  inhibited  it  in  every  series  in  the  experi- 
ment by  at  least  two  dilutions.  In  this  case  we  have  a  cerebrospinal 
fluid  and  an  aqueous  humor  which  contained  agglutinins  for  rabbit 
corpuscles  but  in  these  fluids  the  addition  of  rabbit  serum  completely 
inhibited  the  action.  We  would  not  give  the  impression  that  this 
occurred  normally  in  our  work,  or  even  frequently,  for  in  the  nine 
cases  where  complement  was  used  with  body  fluids  of  immune  dogs, 
this  phenomenon  was  noted  in  only  this  one  experiment.  It  appeared 
only  once  in  the  fluids  of  normal  dogs,  and  in  that  case  the  action  was 
less  marked. 

Conclusions. — i .  In  the  blood  of  dogs  immunized  with  alien  blood 
hemolysins  are  faund  in  the  serum,  thoracic  lymph,  and  neck  lymph, 
and  usually  in  the  pericardial  fluid.  They  are  not  found  in  the  cere- 
brospinal fluid  or  aqueous  humor.  The  comparative  concentration 
is  the  same  as  in  the  normal  animal. 

2.  -The  addition  of  guinea-pig  serum  as  complement  in  non- 
hemolytic  doses  increases  greatly  the  hemolytic  power  of  the  serum, 
neck  lymph,  thoracic  lymph,  and  pericardial  fluid;   therefore,  in  the 
course  of  immunization  the  amboceptors  are  increased  in  the  fluids, 
while  complement  is  not.     Cerebrospinal  fluid  and  aqueous  humor 
do  not  become  hemolytic  on  the  addition  of  complement;   therefore, 
they  do  not  contain  amboceptors. 

3.  In  the  immunized  dog  the  agglutinins  are  more  concentrated 
than  in  the  same  fluids  of  the  normal  animal.     The  usual  order  of 
descending  concentration  is:    serum,  thoracic  lymph,  neck  lymph, 
pericardial  fluid;   but  the  order  may  be  thoracic  lymph,  serum,  neck 
lymph,  pericardial  fluid.     Cerebrospinal  fluid  and  aqueous  humor 
may  have  agglutinins  present  but  usually  do  not.     If  agglutinins 
are  present  in  these  two  fluids,  the  concentration  is  about  equal  and 
lower  than  the  pericardial  fluid. 

4.  Immunization  of  a  dog  to  horse  serum  increases  the  hemolytic 
power  of  the  body  fluids  for  horse  corpuscles,  but  little  if  at  all  for 
rabbit  corpuscle.     The   hemagglutinins   are   increased   to   a   slight 
extent. 

5.  Occasionally   the  addition  of  rabbit  serum  will  inhibit  the 
agglutination  of  washed  rabbit  corpuscles  by  the  fluids  of  a  dog 
immune  to  rabbit  blood. 


140  FRANK  C.  BECHT  AND  JAMES  R.  GREER 

PROTEIN   PRECIPITINS. 

Since  most  investigators  who  have  worked  with  precipitins  are 
agreed  as  to  the  delicacy  and  specificity  of  the  reaction,  we  chose 
them  as  one  of  the  antibodies  best  suited  for  study  in  our  work  on  the 
body  fluids  of  normal  and  immune  animals.  In  several  cases  these 
were  the  same  animals  used  in  the  work  on  hemolysins  and  hemag- 
glutinins. 

Our  method  was  the  same  as  is  usually  employed,  namely,  a 
dilution  method.  Doses  of  the  immune  fluid  varying  between  o .  2  c.c. 
and  o.oi  c.c.  were  placed  in  a  series  of  test  tubes  and  made  up  to 
2  c.c.  with  sterile  0.9  per  cent  Nad  solution.  To  these  tubes  were 
then  added  0.15  c.c.  of  the  same  serum  as  used  for  immunization. 
Control  experiments  were  made  in  case  of  each  of  the  fluids,  and  of 
the  serum,  to  eliminate  any  possibilities  of  a  sediment  from  the  protein 
solutions  confusing  the  results.  The  tubes  were  incubated  for  two 
hours  at  37°  C.,  and  then  left  in  the  ice-box  12  to  20  hours  before  the 
final  reading  was  made. 

Our  results  were  as  follows:  In  the  fluids  of  three  normal  dogs 
tested  with  the  fresh  serum  of  the  rabbit  not  a  trace  of  precipitate 
appeared  in  any  tube. 

In  experiments  with  the  fluids  of  seven  dogs  immune  to  rabbit 
blood,  three  gave  positive  and  four  negative  results.  One  dog  gave 
a  precipitate  only  in  the  first  dilution  of  the  serum  (i :  10) ;  the  other 
mixtures  and  the  control  mixtures  remained  perfectly  clear.  This 
animal  had  been  immunized  by  repeated  injections  of  rabbit  blood 
intraperitoneally,  receiving  in  all  57  c.c.  between  November  14 
and  December  5,  1908. 

A  dog  which  10  days  earlier  had  received  an  intraperitoneal 
injection  of  150  c.c.  of  rabbit  blood  gave  a  positive  reaction  in 
both  serum  and  thoracic  lymph.  In  this  case  the  precipitation 
occurred  in  a  much  higher  dilution  than  in  the  former,  tho  in  the 
latter,  neither  the  neck  lymph,  pericardial  fluid,  cerebrospinal  fluid, 
nor  the  aqueous  humor  nor  any  of  the  controls  showed  any  pre- 
cipitate. 

A  dog  immunized  by  the  intraperitoneal  injection  of  80  c.c.  of 
rabbit  blood  gave  the  best  results  of  all,  the  serum  and  thoracic 
lymph  giving  precipitation  in  dilutions  of  i :  40  and  the  <neck  lymph 


CONCENTRATION  OF  ANTIBODIES  141 

in  i :  20 ;  the  pericardial  and  cerebrospinal  fluids  and  the  aqueous 
humor,  however,  gave  no  reaction  in  i :  10. 

Of  the  five  attempts  to  produce  precipitins  in  dogs  by  immuniza- 
tion with  horse  serum,  four  were  entirely  negative,  and  even  in  the  one 
positive  result  only  the  serum,  thoracic  lymph,  and  the  neck  lymph 
contained  precipitin.  The  precipitation  occurred  in  a  higher  dilution 
in  the  thoracic  lymph  than  in  the  serum,  but  only  in  the  lower  dilu- 
tions of  the  neck  lymph,  which  was  considerably  weaker  than  the 
serum. 

Our  data  are  not  uniform  enough  nor  extensive  enough  to  warrant 
us  in  drawing  conclusions.  It  is  apparent  that  dogs  develop  pre- 
cipitins with  extreme  difficulty,  and  that  a  good  method  of  immuniza- 
tion is  by  single,  large,  intraperitoneal  injections.  The  results  that 
we  have  seem  to  show  that  the  precipitins  follow  closely  the  hemag- 
glutinins  and  the  hemolysins  in  their  distribution  in  the  various  body 
fluids  of  immune  animals,  altho  we  have  not  as  yet  been  able  to 
demonstrate  any  in  the  pericardial  fluid. 

BACTERIAL   AGGLUTININS. 

We  also  made  a  study  of  the  concentration  of  the  agglutinins  for 
the  typhoid  bacillus  in  the  various  body  fluids  of  normal  and  immu- 
nized cats  and  dogs,  and  intend  later  to  extend  the  work  to  cover 
the  bacteriolysins. 

Nuttall,34  employing  both  the  hanging  drop  method  and  the  plate  method,  found 
bacteriolysins  for  the  anthrax  bacillus  in  the  aqueous  humor  and  the  pleuritic  exudate 
of  dogs  and  rabbits.  Prudden38  found  bacteriolysins  in  the  amniotic,  hydrocele,  and 
acites  fluids.  Meltzer  and  Norris28  found  the  thoracic  lymph  nearly  as  bacteriolytic  as 
the  serum  for  the  typhoid  bacillus.  Widal43  found  agglutinins  for  the  typhoid  bacillus 
active  in  a  dilution  of  i :  60  in  the  pericardial  fluid,  while  the  serum  of  the  same  patient 
was  active  at  1:350.  Edema  fluid  was  found  strongly  agglutinating  also,  but  the 
result  with  cerebrospinal  fluid  was  negative.  Picks?  found  that  cerebrospinal  fluid 
agglutinated  at  a  dilution  of  i :  i  and  i :  2.  Kohler20  found  agglutinins  in  the  cerebro- 
spinal fluid  in  one  case  in  ten  examined  of  non-typhoid  patients,  and  but  three  times 
out  of  19  cases  of  typhoid  fever,  and  these  in  dilutions  of  i :  i,  1:5,  and  i :  10.  Braude 
and  Carlson?  made  a  study  of  the  concentration  of  the  agglutinins  for  the  typhoid 
bacillus  in  the  body  fluids  of  normal  and  immunized  cats  and  dogs.  Inasmuch  as  they 
used  for  comparison  the  action  of  the  fluids  of  the  same  animal  upon  the  same  bacterial 
suspension,  their  results  give  more  nearly  the  true  conditions  of  the  body  fluids  in  an 
animal;  they  avoided  the  error  which  a  comparison  of  the  body  fluids  of  different 
animals  is  sure  to  introduce  on  account  of  the  wide  individual  variations.  The  hanging 
drop  method  which  they  employed,  however,  is  not  considered  as  accurate  as  the  pre- 


142 


FRANK  C.  BECHT  AND  JAMES  R    GREER 


cipitation  method  which  we  used.     They  found  bacterial  precipitins  in  the  cerebrospinal 
fluid  of  immunized  dogs  and  cats. 

The  fluids  used  in  these  experiments  were  collected  as  described 
at  the  beginning  of  this  paper.  The  test  tubes  were  the  same  as 
those  used  in  the  hemolytic  work.  The  bacteria  were  secured  from 
2o-24-hour  slant  agar  cultures,  made  up  with  sterile  0.9  per  cent 
NaCl  solution  to  a  distinctly  cloudy  suspension,  and  then  filtered 
through  filter  paper  to  remove  all  of  the  clumps.  A  series  of  tubes 
was  arranged  for  each  fluid,  then  after  the  proper  amount  of  fluid  had 
been  measured  into  each,  the  bacterial  suspension  was  added.  The 
dilutions  used  were  i :  10,  i :  50,  i :  100,  i :  500,  i :  2,000,  i :  6,000.  Our 
lowest  dilution  was,  perhaps,  too  high  to  detect  the  traces  of  agglutinins 
reported  by  Pick  and  Kohler.  The  tubes  were  all  incubated  together 
at  37°  C.  for  two  hours  and  then  kept  in  the  ice-box  for  from  12-20 
hours  before  the  final  observations. 

In  our  study  of  agglutinins  for  the  typhoid  bacillus  we  used  normal 
cats  and  dogs,  animals  actively  immunized  by  the  repeated  injection 
of  typhoid  cultures,  and  animals  rendered  passively  immune  by  the 
withdrawal  of  large  quantities  of  blood  from  the  normal  animal,  and 
the  subsequent  injection  of  an  equal  amount  of  warm,  defibrinated 
blood  from  an  actively  immunized  animal  of  the  same  kind. 

A.  Normal  animals. — We  studied  first  the  concentration  of 
agglutinins  in  the  body  fluids  of  normal  cats  and  found  agglutinins 
for  the  typhoid  bacillus  active  in  a  dilution  from  i :  10  in  the  serum 
and  the  thoracic  lymph;  the  neck  lymph  and  the  pericardial  fluid 
usually  contain  them  in  the  same  concentration  but  the  cerebrospinal 
fluid  and  the  aqueous  humor  do  not  contain  them  in  such  dilution. 

DOGS. — A  study  of  the  concentration  of  the  typhoid  agglutinins 
in  normal  dogs  gave  the  results  shown  in  the  following  table : 

TABLE  6. 

THE  COMPARATIVE  AGGLUTINATING  POWER  ON  THE  TYPHOID  BACILLUS  OF  THE  BODY  FLUIDS  OF  A 

NORMAL  DOG. 


Dilution 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericardial 
Fluid 

Cerebro- 
spinal Fluid 

Aqueous 
Humor 

i  ;  10  

+  + 

+  + 

+  + 

0 

o 

o 

1:50  

+  + 
+ 

0 

0 

o 

o 
o 

0 

o 

0 
0 

o 

o 

o 

0 

0 

0 

CONCENTRATION  OF  ANTIBODIES 


This  experiment  is  one  of  four  performed  on  normal  animals,  and 
gives  a  fair  idea  of  the  concentration  of  the  agglutinins  in  the  body 
fluids.  There  are,  of  course,  variations  between  animals  even  in 
the  same  species.  In  all  of  our  experiments  the  concentration  of 
agglutinins  was  highest  in  the  serum,  less  in  the  thoracic  and  the 
neck  lymph,  and  the  pericardial  fluid  contained  agglutinins  in  only 
one  case.  The  cerebrospinal  fluid  and  the  aqueous  humor  did  not 
contain  any  in  any  case. 

B.  Actively  immunized  animals. — We  made  a  study  of  the  fluids 
of  five  immunized  cats.  Three  had  received  repeated  subcutaneous 
injections  of  typhoid  bacilli.  For  the  first  few  injections  killed  cul- 
tures, but  later  live  cultures  were  used.  The  doses  injected  were 
increased  gradually  and  careful  record  of  the  weight  and  general 
condition  kept,  to  guard  against  pushing  the  process  too  rapidly. 
The  remaining  two  animals  received  a  single  large  injection  of  six 
living  24-hour  cultures.  The  results  given  in  Table  7  may  be  taken 
as  fairly  typical. 

TABLE  7. 
THE  COMPARATIVE  AGGLUTINATING  POWER  ON  THE  TYPHOID  BACILLUS  OF  THE  BODY  FLUIDS  OF  AN 

IMMUNIZED  CAT. 

(Immunized  by  repeated  subcutaneous  injections.     Fluids  collected  five 
days  after  the  last  injection.) 


Dilution 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericardial 
Fluid 

Cerebro- 
spinal Fluid 

Aqueous 
Humor 

:io..  
:  50  

+  + 
+  + 

+  4- 
+  + 

+  + 

+  + 

+ 
o 

? 
o 

? 
o 

:ioo  
:  500  

+  + 

+  + 
o 

+  + 
+ 

o 
o 

o 
o 

0 

o 

-.2,000  
:  6,000  

+ 
+ 

0 
0 

o 

0 

0 
0 

0 
0 

0 

o 

Control=o. 

The  other  experiments  showed  some  variations.  In  two  cases  in 
five  the  agglutinins  were  as  concentrated  in  the  thoracic  lymph  as  in 
the  serum,  in  the  remaining  three  of  five  the  serum  was  by  far  the 
more  concentrated  of  the  two.  In  one  case  the  neck  lymph  showed 
the  same  concentration  of  agglutinins  as  the  thoracic  lymph,  but  both 
were  considerably  lower  than  the  serum;  in  the  four  remaining  cases 
the  concentration  in  the  neck  lymph  was  lower  than  in  the  thoracic 
lymph.  Agglutinins  were  found  in  four  of  five  cases  in  the  peri- 
cardial fluid,  but  in  no  case  in  a  dilution  higher  than  i :  10.  Agglu- 
tination in  the  fifth  case  was  questioned.  The  cerebrospinal  fluid 


144 


FRANK  C.  BECHT  AND  JAMES  R.  GREER 


was  negative  in  all  dilutions  used  in  four  of  five  cases,  and  agglutina- 
tion in  the  fifth  case  at  i :  10  was  questioned.  The  aqueous  humor 
gave  negative  results  in  three  of  five  cases,  and  the  agglutination  was 
questioned  in  the  remaining  two  at  a  dilution  of  i :  10. 

The  development  of  agglutinins  for  the  typhoid  bacillus  in  dogs 
runs  a  course  which  is  strictly  comparable  to  that  in  the  cats  under 
similar  circumstances. 

TABLE  8. 
THE  COMPARATIVE  AGGLUTINATING  POWER  FOR  THE  TYPHOID  BACILLUS  OF  THE  BODY  FLUIDS  OF  AN 

IMMUNE   DOG. 
(Immunized  by  repeated  subcutaneous  injections.) 


Dilution 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericardia! 
Fluid 

Cerebro- 
spinal 
Fluid 

Aqueous 
Humor 

Control 

:  10  
•.50  

+  + 
+  + 

+  + 

+  + 

+  + 
+  + 

+  + 
+  + 

0 

0 

0 

:  100  

+  + 

+  + 

+  + 

+ 

:  500  .  .  . 

+  + 

4. 

+  + 

:  2,000  
:  6,000. 

+ 

0 

+ 

o 

From  Table  8  it  is  evident  that  the  concentration  of  the  agglutinins 
in  the  body  fluids  of  immune  dogs  runs  a  course  parallel  to  that  in  the 
immune  cats.  The  concentration  in  the  neck  lymph  and  the  peri- 
cardial  fluid  is  considerably  lower  than  that  in  the  thoracic  lymph 
and  serum,  and  of  the  two  the  concentration  in  the  neck  lymph  is  the 
higher.  In  our  seven  experiments  on  typhoid  immune  dogs,  thoracic 
lymph  and  serum  showed  the  same  concentration  of  bacterial  agglu- 
tinins in  four  cases;  in  the  remaining  three  cases  the  concentration  is 
greater  in  the  serum.  The  pericardial  fluid  contained  agglutinins 
in  six  of  seven  cases.  The  highest  dilution  at  which  agglutination 
occurred  was  i :  100  (three  cases) .  In  no  case  were  agglutinins 
found  in  the  cerebrospinal  fluid  in  the  dilutions  used.  Traces  of 
agglutinins  were  observed  in  three  of  six  cases  in  the  aqueous  humor 
in  a  dilution  of  i :  10. 

We  considered  it  of  interest  to  determine  whether  immunity  to 
rabbit  blood  affected  in  any  way  the  agglutinins  for  the  typhoid 
bacillus  in  the  body  fluids  of  a  dog.  With  this  in  mind  we  tested  the 
usual  six  fluids  of  one  of  our  immune  dogs.  An  increased  agglutinat- 
ing power  could  be  expected,  if,  in  terms  of  Ehrlich's  hypothesis, 
there  were  any  cell  receptors  common  both  to  blood  cells  and  the 
typhoid  bacilli. 


CONCENTRATION  OF  ANTIBODIES 


TABLE  9. 
THE  COMPARATIVE  AGGLUTINATING  POWER  ON  THE  TYPHOID  BACILLUS  OF  THE  BODY  FLUIDS  OF  A 

DOG  IMMUNIZED  WITH  RABBIT  BLOOD. 

(Intraperitoneal  injections  as  follows:  October  10,  ioc.c.;  October  19,  8  c.c.;  October  24,  8  c.c.; 
October  29,  9  c.c.;  November  3,  10  c.c.;  November  9,  10  c.c.;  November  14,  10  c.c.;  fluids  drawn 
November  24.) 


Dilution 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericardial 
Fluid 

Cerebro- 
spinal  Fluid 

Aqueous 
Humor 

+  + 

+ 

4- 

o 

o 

o 

I'-So  
i  :  100  
i  :  500  

+ 
+ 
o 

o 

0 
0 

+ 
o 

0 

0 

o 

0 

0 

o 

0 

0 

o 

0 

A  comparison  of  this  table  with  Table  7  shows  that  the  agglutinins 
for  the  typhoid  bacillus  were  little  if  any  higher  than  those  of  the 
normal  animal,  altho  this  was  one  of  our  most  highly  immunized 
animals  and  agglutinated  rabbit  corpuscles  strongly  in  a  dilution  of 
i :  1,536  in  the  serum  and  in  a  dilution  of  1 1384  in  the  neck  and  the 
thoracic  lymph.  The  relative  concentration  in  the  body  fluids  is 
strictly  comparable  to  those  in  the  normal  animal  cited.  We  tested 
also  the  blood  serum  of  two  other  dogs  immune  to  rabbit  blood  with 
exactly  similar  results. 

C.  Passive  immunity. — Evidence  that  antibodies  of  various  kinds 
are  able  to  pass  through  membranes  is  not  lacking  in  the  literature. 
Ehrlich10  found  that  the  young  from  a  mouse  immune  to  abrin,  ricin, 
or  robin  possess  an  immunity  to  these  poisons  which  persists  for  two 
months.  Ascoli1  found  that  the  antibodies  of  the  new  born  child 
come  from  the  maternal  circulation  and  are  not  formed  in  the  fetus 
itself.  This  being  true,  the  antibodies  of  the  fetus  have  penetrated 
the  walls  of  at  least  a  double  membrane.  Merkle29  found  that  the 
same  was  true  for  rabbits,  since  the  young  born  of  a  mother  immune 
to  human  blood  contain  antibodies  for  human  blood,  altho  they  suckle 
a  normal  mother  from  the  very  first.  Liidke23  confirmed  the  work 
of  Merkle.  Ricketts39  has  shown  that  the  young  born  to  a  guinea- 
pig  mother  immune  to  Rocky  Mountain  spotted  fever  possess  an 
immunity  to  that  disease,  altho  they  suckled  a  normal  mother. 
DeBlasi8  has  shown  that  if  a  cat  is  immunized  post  par  turn  to  B. 
dysenteriae  the  young  develop  an  immunity  from  the  milk.  This  is 
even  a  more  striking  example  than  the  others  of  the  ability  of  anti- 
bodies to  penetrate  membranes,  for  in  this  case  the  antibodies  after 
reaching  the  alimentary  canal  of  the  young  animal  must  penetrate 


146 


FRANK  C.  BECHT  AND  JAMES  R.  GREER 


the  intestinal  mucosa,  and  later  the  capillary  or  lymphatic  wall, 
depending  upon  whether  the  path  of  absorption  was  the  blood  or 
the  lymph. 

It  was  hoped  that  a  careful  study  of  the  passage  of  these  antibodies 
from  the  blood  into  the  various  body  fluids  in  a  passively  immune 
animal  would  throw  some  light  upon  the  problem  of  lymph  formation. 
We  have  been  disappointed  thus  far,  for  our  results  have  not  been 
decisive  enough  to  warrant  any  general  conclusions. 

Passive  immunity  was  established  by  the  withdrawal  under  light 
ether  anesthesia  in  most  cases  of  a  large  amount  of  blood  from  a 
normal  animal,  and  the  subsequent  injection  of  an  equal  amount  of 
warm,  defibrinated  blood  from  an  actively  immune  animal  of  the  same 
kind. 

CATS. — To  show  the  concentration  of  agglutinins  in  the  body  fluids 
of  a  passively  immune  cat  we  give  Table  10  as  typical  of  our  results. 
It  will  also  show  the  concentration  of  agglutinins  of  the  actively 
immune  cat  from  which  the  blood  was  taken. 

TABLE    io. 
THE  COMPARATIVE  AGGLUTINATING  POWER  ON  THE  TYPHOID  BACILLUS  OF  THE  BODY  FLUIDS  OF  CATS 

ACTIVELY  AND  PASSIVELY  IMMUNE  TO  THE  TYPHOID  BACILLUS. 

(Cat  n,  immunized  June  29  by  the  injection  subcutaneously  of  six  live  24-hour  slant  agar  cultures 
of  the  typhoid  bacillus,  operated  July  9.  Cat  12,  passively  immunized  by  the  withdrawal  on  July  9  of 
100  c.c.  of  blood  and  the  injection  of  100  c.c.  of  blood  from  Cat  n.  Operated  July  io.) 


DILUTION 

SERUM 

NECK 
LYMPH 

THORACIC 
LYMPH 

PERICAR- 

DIAL 

FLUID 

CEREBRO- 

SPINAL 

FLUID 

AQUEOUS 
HUMOR 

ii 

12* 

12 

ii 

12 

n 

12 

ii 

12 

ii 

12 

ii 

12 

:io  
150  

+  + 
+  + 
+  + 
+ 
+ 

0 

+ 
+ 
0 
0 
0 
0 

+  + 
+ 

+ 
+ 
0 
0 

+  + 
+  + 
+ 
+ 
+ 

0 

+  + 
+ 
0 
0 
0 
0 

+  + 
+  + 
+ 
+ 
+ 

0 

+  + 
+ 
+ 
0 

o 

0 

+ 
+ 

0 
0 

o 

0 

O 
0 
0 
0 
0 
0 

o 

0 

o 

0 

o 

0 

O 
O 

o 

0 

o 

0 

o 

0 

o 

0 
0 
0 

O 
0 
0 
0 
0 
0 

:ioo  

12,000  

:  6,000  

*  Normal  serum  before  injection  of  immune  serum  1 1 . 

From  Table  io  it  is  evident  that  it  is  possible  to  increase  the  agglu- 
tinins not  only  in  the  serum  but  in  both  of  the  lymphs  by  a  process  of 
passive  immunization.  The  relative  concentration  is  maintained 
in  the  fluids  which  we  find  in  the  actively  immunized  animals  of  the 
same  grade  of  immunity.  In  no  case  in  the  passively  immunized 
animal  did  the  pericardial  fluid  show  any  increase  over  the  normal 
animal.  The  cerebrospinal  fluid  and  the  aqueous  humor^  showed  no 


CONCENTRATION  or  ANTIBODIES  147 

increase,  and,  indeed,  one  would  not  expect  that  they  would,  since 
in  the  actively  immune  animal  the  presence  of  antibodies  is  the  excep- 
tion and  not  the  rule.  The  results  here  are  typical  of  all  of  our  results. 
In  four  passively  immune  cats  this  same  increase  in  the  concentration 
of  the  agglutinins  in  serum,  neck  lymph,  and  the  thoracic  lymph  was 
noted.  In  two  of  the  four  cases  the  concentration  in  the  neck  lymph 
and  the  thoracic  lymph  was  equal,  in  the  remaining  two  of  the  four 
experiments  the  concentration  in  the  thoracic  lymph  was  greater 
than  that  in  the  neck  lymph.  There  appears  on  the  whole  to  be  a 
tendency  for  the  concentration  of  the  agglutinins  in  the  thoracic  and 
the  neck  lymph  in  the  passively  immune  animals  to  run  more  nearly 
parallel  than  in  the  actively  immunized  animals. 

DOGS. — Passive  immunity  produced  in  dogs  in  the  manner 
described  yields  exactly  similar  results;  namely,  the  concentration  in 
agglutinins  of  the  serum,  neck  lymph,  and  thoracic  lymph  can  be 
greatly  increased.  The  concentration  of  the  agglutinins  in  the  peri- 
cardial  fluid,  cerebrospinal  fluid,  and  the  aqueous  humor  is  no  higher 
than  in  the  normal  animal. 

In  one  of  three  passively  immunized  dogs  the  concentration  in  the 
neck  lymph  and  thoracic  lymph  was  equal;  in  the  other  two  the 
thoracic  lymph  was  much  higher.  The  fact  that  50  per  cent  of  our 
passively  immunized  cats  and  33  per  cent  of  the  passively  immune 
dogs  showed  an  equal  concentration  of  the  agglutinins  in  the  thoracic 
and  the  neck  lymph  may  be  significant,  for  the  percentage  of 
such  findings  in  the  actively  immune  animals  is  14  per  cent  for 
the  dogs  and  16  per  cent  for  the  cats;  but  much  more  extensive 
experimentation  is  necessary  before  any  reliance  can  be  placed  on 
these  results. 

In  order  to  determine  the  relative  rapidity  with  which  these  anti- 
bodies pass  from  the  blood  into  the  lymph  the  following  experiment 
was  made.  A  dog  was  anesthetized  and  rendered  passively  immune 
by  the  withdrawal  of  300  c.c.  of  blood  from  the  femoral  artery  and 
the  injection  of  300  c.c.  of  blood  and  serum  from  a  typhoid  immune 
dog.  Small  samples  of  blood  were  drawn  at  stated  intervals,  as 
were  also  samples  of  neck  lymph.  Thoracic  lymph,  pericardial 
fluid,  cerebrospinal  fluid,  and  aqueous  humor  were  collected  at  the 
end  of  the  experiment.  All  of  the  fluids  except  the  thoracic  lymph 


148      FRANK  C.  BECHT  AND  JAMES  R.  GREER 

which  was  tinged  with  blood  were  in  good  condition  and  were  tested 
in  the  usual  manner.     The  results  are  shown  in  Table  1 1 . 

TABLE  ii. 

THE  RATE  OF  PASSAGE  OF  ANTIBODIES  FROM  THE  BLOOD  INTO  THE  BODY  FLUIDS  IN  PASSIVELY 

IMMUNE  ANIMALS. 

Highest  Dilu- 

Fluid  tion  Showing 

Agglutination 


Normal  serum 

Immune  serum  injected  into  passively  immune  dog. . 

Equal  parts  of  normal  and  immune  sera 

Serum  25  min.  after  transfusion 

"       i  hr.  25  min.  after  transfusion 

"       2    "     "      "        "  "          

14       3    "     "      "        "  "          

"       4    "     "       "         "  " 

Neck  lymph  3  hr.          "  "          


4    "    30  min.  after  transfusion. . 


:  100 
:  2,000 
:  2,000 

:5°° 
•.500 
:  500 
=  500 


'•5° 


Thoracic  lymph  4  hr.  30  min.  after  transfusion i :  100 

Pericardial  fluid  4    "    30     "        "  o 

Cerebrospinal  fluid o 

Aqueous  humor o 

It  may  be  noted  that  the  results  of  this  experiment  are  practically 
identical  with  those  secured  from  our  24-hour  experiments  on  the 
passively  immunized  dogs.  The  concentration  of  the  agglutinins  of 
the  serum  remain  practically  the  same  during  the  4^  hours  of  this 
experiment.  The  neck  lymph  had  the  same  concentration  of  agglu- 
tinins three  hours  after  the  transfusion  that  it  had  at  the  close  of  the 
experiment.  The  thoracic  lymph  in  this  case  showed  considerably 
higher  than  the  neck  lymph,  part  of  which  may  have  been  due  to  the 
serum  in  the  lymph,  which  came  either  through  the  increased  permea- 
bility of  the  capillary  walls  due  to  the  action  of  the  anesthetic  or  to 
trauma,  or  to  the  natural  anastomoses  between  the  lymphatics  and 
the  blood  vessels  of  the  splanchnic  area. 

The  above  experiment  seems  to  indicate  that  the  passage  of  such 
substances  as  bacterial  agglutinins  from  the  blood  to  the  lymphs  is 
a  relatively  rapid  process,  for  the  concentration  of  these  bodies  was 
the  same  in  the  body  fluids  in  4^  hours  as  in  24  hours  after  passive 
immunization. 

From  the  results  obtained  the  following  conclusions  seem  justified: 

i.  Agglutinins  for  the  typhoid  bacillus  are  found  in  the  serum  and 
thoracic  lymph  of  normal  cats  in  approximately  equal  amounts. 


•W*a       4 

i: 

CONCENTRATION  OF  ANTIBODIES  149 

They  may  or  may  not  be  present  in  the  neck  lymph  and  the  pericardial 
fluid.  They  are  not  found  in  the  cerebrospinal  fluid  nor  the  aqueous 
humor. 

2.  Agglutinins  for  the  typhoid  bacillus  are  found  in  the  serum, 
neck  lymph,  and  the  thoracic  lymph  in  normal  dogs  in  relatively 
higher  concentrations  than  in  the  same  fluids  in  normal  cats.     They 
are  most  concentrated  in  the  serum,  are  in  nearly  equal  concentration 
in  the  two  lymphs.    They  may  or  may  not  be  found  in  the  pericardial 
fluid.     They  are  found  neither  in  the  cerebrospinal  fluid  nor  the 
aqueous  humor. 

3.  Agglutinins  for  typhoid  bacilli  are  found  in  actively  immune 
cats  in  the  serum,   thoracic  lymph,   neck  lymph,   and  pericardial 
fluid  in  decreasing  concentration  in  the  order  mentioned.     If  found 
in  the  cerebrospinal  fluid  and  aqueous  humor  there  are  only  traces. 

4.  Agglutinins  for  typhoid  bacilli  are  found  in  actively  immune 
dogs  in  the  serum,   thoracic  lymph,  neck  lymph,  and  pericardial 
fluid,  usually  in  decreasing  concentration  in  the  order  named.     Serum 
and  thoracic  lymph  may  show  an  equal  concentration.     Cerebrospinal 
fluid  shows  no  agglutinins  in  a  dilution  of  i :  10.     Aqueous  humor 
may  or  may  not  show  agglutinins  in  a  dilution  of  i :  10. 

5.  Immunization  of  a  dog  to  rabbit  blood  does  not  increase  the 
concentration  of  agglutinins  for  typhoid  bacilli  in  the  body  fluids 
above  the  normal. 

6.  In  the  passively  immunized  animal  the  agglutinins  for  typhoid 
bacilli  pass  readily  from  the  blood  stream  into  the  lymphs,  usually  in 
greater  concentration  into  the  thoracic  lymph  than  into  the  neck 
lymph,  but  the  concentration  may  be  equal  in  the  two,  showing  the 
permeability  of  the  two  systems  to  be  the  same.     They  do  not  pass 
into  the  pericardial  fluid,  cerebrospinal  fluid,  nor  the  aqueous  humor. 

7.  The  passage  of  the  agglutinins  from  the  serum  to  the  lymphs 
in  the  passively  immunized  animal  is  a  relatively  rapid  process,  and 
no  difference  is  shown  between  the  concentration  at  the  end  of  4^ 
hours  and  24  hours. 

OPSONINS. 

We  made  a  quantitative  study  of  the  bacterial  opsonins  and  the 
hemopsonins  in  the  body  fluids  of  normal  and  immunized  dogs. 


150  FRANK  C.  BECHT  AND  JAMES  R.  GREER 

Since  the  discovery  of  the  opsonins  by  Wright  and  Douglas44  there  has  been  a 
great  deal  of  work  done  with  these  bodies,  and  as  a  result  there  has  arisen  an  extensive 
and  conflicting  literature.  It  is  beyond  the  scope  of  this  paper  to  undertake  a  general 
discussion  of  this  subject.  We  will  confine  ourselves,  therefore,  to  those  results  which 
most  nearly  relate  to  our  problem.  So  far  as  we  have  been  able  to  determine,  there  has 
been  up  to  this  time  no  study  of  the  relative  concentration  of  the  opsonins  in  the  different 
body  fluids  of  the  same  animal. 

Wright  and  Reid45  found  that  exudates  may  contain  little  or  no  opsonin.  Opiess 
found  that  the  exudates  following  the  injection  of  bacteria  or  turpentine  were  practically 
opsonin  free  if  unmixt  with  blood.  Bohme6  reports  15  cases  of  pleural  and  peritoneal 
exudates  showing  a  concentration  of  the  opsonins  ranging  from  less  than  half  to  even  a 
greater  concentration  than  that  of  the  serum.  He  also  examined  the  edema  fluid  in 
seven  persons.  In  two  of  these  cases  there  was  very  little  opsonin  while  the  remaining 
five  showed  a  considerable  amount,  but  in  no  case  was  it  equal  to  that  of  the  serum. 
This  same  investigator  examined  the  cerebrospinal  fluid  in  16  cases  and  obtained  opsonic 
indices  varying  from  8  per  cent  to  76  per  cent.  He  notes  that  these  fluids  were  free  from 
blood.  Bohme  tries  to  correlate  the  concentration  of  the  opsonin  in  these  cases  with 
the  amount  of  the  protein  in  the  fluid. 

Levaditi  and  Inman21  and  others  have  shown  that  while  the  aqueous  humor 
normally  contains  little  or  no  opsonin,  yet  that  secured  after  a  previous  withdrawal 
contains  the  opsonin. 

Methods. — The  method  of  securing  the  fluids  was  the  same  as  that  described  in 
the  foregoing. 

The  leukocytes  were  from  the  pleural  exudate  of  young,  healthy  dogs  which  24 
hours  previously  had  been  given  intraplural  injection  of  a  suspension  of  aleuronaut 
in  sterile  0.9  per  cent  NaCl  solution.  The  exudate  was  secured  after  bleeding  the 
animal  to  death,  and  was  drawn  into  a  warm  solution  of  i  per  cent  sodium  citrate  in 
0.9  per  cent  NaCl.  The  leukocytes  were  centrifugated  out,  and  washed  a  second  time 
in  warm,  sterile  salt  solution. 

The  bacterial  emulsion  was  obtained  by  suspending  in  0.9  per  cent  NaCl  solution 
24-hour  slant  agar  cultures  of  Staph.  aureus.  This  emulsion  was  filtered  through 
absorbent  cotton  in  order  to  remove  the  clumps  and  was  then  made  up  to  the  desired 
opalescence.  In  all  of  the  experiments  a  fairly  rich  suspension  of  the  bacteria  was 
used,  but  no  attempt  was  made  to  standardize  this  emulsion,  and  comparison  of 
individual  experiments,  except  where  the  same  suspension  of  leukocytes  and  bacteria 
were  used,  would  not  be  warranted. 

The  technic  was  essentially  that  described  by  Walker  with  slight  modification. 
This  method  in  our  hands  gave  the  most  satisfactory  results.  Dilutions  of  the  various 
body  fluids  were  used  in  the  same  way  as  the  whole  fluid.  The  usual  time  for  incuba- 
tion was  20  minutes.  Care  was  taken  to  make  the  smear  from  the  incubated  mixture 
as  soon  after  the  period  of  incubation  as  possible,  so  that  the  time  factor  was  as  nearly 
equal  as  it  could  be  kept.  We  here  incur  a  slight  source  of  error  in  the  difference  in 
the  age  of  the  material,  for  BeattieS  has  shown  that  the  older  citrated  leukocytes  are 
up  to  24  hours,  the  greater  the  phagocytosis,  but  the  maximum  difference  in  time 
between  the  first  and  last  smear  was  never  greater  than  one  hour.  Great  care  was 
taken  to  keep  the  leukocytic  suspension  of  the  same  consistency  throughout  the  experi- 
ment. The  leukocytes  were  used  as  fresh  as  possible,  usually  in  about  two  hours  or 
less  after  removal  from  the  animal's  body.  The  number  of  leukocytes  varied  from 


CONCENTRATION  OF  ANTIBODIES 


60  to  100  for  each  dilution,  but  the  number  was  kept  constant  for  each  experiment. 
The  stains  used  were  carbol-thionin  and  Giemsa's  blood  stain,  but  the  same  stain  was 
used  throughout  the  same  series  of  fluids. 

Inasmuch  as  there  has  been  so  much  criticism  of  the  opsonic  methods  we  made 
the  following  tests  of  the  accuracy  of  our  observations.  Using  the  same  fluids,  leuko- 
cytes, and  bacterial  suspension,  duplicate  tests  were  made.  The  slides  were  labeled 
in  such  a  way  that  the  person  making  the  count  had  no  way  of  knowing  what  the  slide 
contained,  thus  eliminating  the  personal  equation.  The  result  of  this  test  was  that 
while  most  of  the  duplicates  agreed  fairly  closely  there  were  cases  that  varied  as  much 
as  25  per  cent,  but  the  majority  of  the  counts  showed  a  much  closer  agreement. 

Bacteriopsonins. — We  give  the  results  of  an  experiment  which  we 
consider  as  typical  so  far  as  concerns  opsonin  for  Staph.  aureus  in 
normal  dogs. 

TABLE  12. 
COMPARATIVE  STUDY  OF  THE  OPSONIN  FOR  STAPH.  AUREUS  IN  THE  BODY  FLUIDS  OF  A  NORMAL  DOG  . 


Dilution 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericardia! 
Fluid 

Cerebro- 
spinal  Fluid 

Aqueous 
Humor 

Whole 

3  4° 

3  5° 

o  81 

i  08 

1:10  

0.30 

0.16 

0.58 

0.32 

Control  =  0.36. 

It  will  be  noted  that  in  this  experiment  the  opsonin  had  practically 
disappeared  in  the  dilution  of  i :  10  in  all  of  the  fluids.  In  the  undi- 
luted fluids  the  concentration  of  the  opsonin  in  the  serum,  neck,  and 
thoracic  lymph  was  nearly  the  same,  but  there  was  a  slight  excess  in 
favor  of  the  thoracic  lymph.  The  pericardial  fluid,  cerebrospinal 
fluid,  and  the  aqueous  humor  contained  opsonin  in  a  very  much  lower 
concentration. 

In  four  of  nine  normal  animals  the  concentration  of  the  opsonin 
in  the  serum  was  considerably  higher  than  in  the  thoracic  and  the 
neck  lymph.  In  three  of  nine  cases  the  concentration  was  practically 
equal  in  the  serum  and  the  two  lymphs.  In  five  of  eight  experiments 
the  amount  of  opsonin  in  the  neck  lymph  was  considerably  less  than 
in  the  serum  and  thoracic  lymph.  It  is  thus  evident,  that,  as  far  as 
these  three  fluids  are  concerned,  the  concentration  is  greatest,  on  the 
average,  in  the  serum,  least  in  the  neck  lymph,  while  the  thoracic 
lymph  occupies  an  intermediate  position. 

Opsonin  was  found  in  the  cerebrospinal  fluid  of  four  of  seven  dogs, 
but  in  every  case  it  was  in  decidedly  smaller  amounts  than  in  the 
serum  and  lymphs  of  the  same  animal.  In  the  other  three  animals 
there  was  no  opsonin  in  this  fluid. 


FRANK  C.  BECHT  AND  JAMES  R.  GREEK 


The  aqueous  humor  contained  opsonin  in  five  of  eight  cases,  but 
in  only  one  of  these  did  the  concentration  approach  that  of  the  serum 
of  the  same  animal.  The  remaining  four  showed  very  much  less 
phagocytosis,  while  three  experiments  gave  negative  results. 

Opsonin  was  found  in  the  pericardial  fluid  in  two  of  seven  dogs 
in  considerable  amounts,  in  one  it  was  practically  equal  to  the  neck 
lymph  of  the  same  animal.  In  one  of  the  remaining  animals  there 
was  a  trace  of  opsonin.  The  other  four  animals  contained  no  opsonin 
in  this  fluid. 

It  is  difficult  to  increase  to  any  marked  extent  the  concentration 
of  the  opsonin  for  Staph.  aureus  in  a  dog  by  immunization. 

We  submit  below  two  experiments  in  detail  which  we  consider 
quite  typical  of  our  results. 

TABLE  13. 
COMPARATIVE  OPSONIC  POWER  FOR  STAPH.  AUREUS  OF  THE  BODY  FLUIDS  OF  IMMUNIZED  DOGS. 

(Both  of  these  dogs  received  subcutaneously  six  slant  agar  cultures  of  Staph.  aureus  suspended  in 
0.9  per  cent  NaCl  solution.  Ten  days  later  the  fluids  were  all  secured  in  apparently  perfect  condition. 
Fluids  kept  over  night  in  ice-box.  Bacteria  from  a  24-hour  slant  agar  growth.  Incubation  20  minutes. 
Carbol-thionin.  Same  bacterial  suspension  and  leukocytes  in  both  experiments.) 


Dilution 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericar- 
dial 
Fluid 

Cerebro- 
spinal 
Fluid 

Aqueous 
Humor 

Dog  I 

Whole 

i  8s 

i  :  10 

4-Si 

i-7S 

3-Si 

i-33 

0.88 

0.50 

1:50 

1-75 

0.86 

i  .01 

0.22 

0.28 

0.28 

Dog  II  

Whole 

10.07 

6.67 

8-'3 

4.18 

0.92 

i-33 

1:10 

4.21 

1.50 

2.88 

1-47 

0.22 

*     0.30 

1:50 

O.po 

0.38 

0.52 

0.17 

0.22 

0-35 

Control  for  both=o.22. 

It  will  be  noted  that  the  results  of  these  experiments  are  com- 
parable with  those  with  respect  to  the  other  antibodies  studied.  In 
the  two  experiments  cited  above  the  concentration  of  the  opsonin  is 
considerably  higher  in  the  serum  than  in  the  other  fluids.  Of  the 
two  lymphs  there  is  a  slight  excess  in  that  from  the  thoracic  duct  over 
that  from  the  cervical  lymphatics.  In  both  of  these  experiments 
while  there  is  considerable  opsonin  in  the  pericardial  fluid  yet  it  is 
less  than  in  the  serum  and  the  lymphs.  In  both  of  these  experiments 
the  cerebrospinal  fluid  and  the  aqueous  humor  contained  opsonin, 
but  in  very  much  smaller  amounts  than  in  the  corresponding  serum 
and  lymphs. 

Conclusions. — There  is  a  considerable  amount  of  opsonin  for 


CONCENTRATION  OF  ANTIBODIES  153 

Staph.  aureus  in  the  body  fluids  of  normal  dogs.  The  amount  found 
in  immune  dogs  is  quite  comparable  with  this,  as  regards  both  the 
amount  and  the  relative  distribution. 

Opsonin  may  be  present  in  considerable  concentration  in  all  of 
the  body  fluids  studied  by  us,  but  in  the  serum,  thoracic,  and  neck 
lymphs  they  are  always  found  in  greater  concentration  than  in  the 
other  fluids  studied.  Of  these  fluids  the  serum  usually  contains  the 
greatest  amount,  and  the  thoracic  lymph  usually  more  than  the  cor- 
responding neck  lymph.  The  pericardial  fluid,  cerebrospinal 
fluid,  and  the  aqueous  humor  may  or  may  not  contain  opsonin,  but 
rarely  in  amounts  comparable  to  those  of  the  serum  and  lymphs. 

HEMOPSONINS. 

It  has  long  been  noted  that  red  corpuscles  were  taken  up  by  phagocytes  under 
certain  conditions  but  it  was  not  until  opsonins  were  discovered  by  Wright  and  Douglas 
that  the  role  of  the  serum  in  phagocytosis  ^vvas  understood.  Neufeld  and  Topfer33 
showed  that  there  was  something  termed  by  them  "hemotropic  substance"  in  the 
serum  of  a  rabbit  immune  to  goat  blood,  which  caused  the  phagocytosis  of  goat  erythro- 
cytes  by  guinea-pig  leukocytes  in  vitro.  Barrats  found  the  same  antibody  in  the  serum 
of  doves  immune  to  hen  blood.  He  proved  these  bodies  thermostable.  Hektoen16 
was  the  first  to  point  out  the  similarity  of  these  substances  to  bacterial  opsonins  and 
suggested  the  name  "hemopsonins."  Neufeld  and  Topfer,  Barrat,  Keith,  and  Hektoen 
all  cite  evidence  to  show  that  these  substances  are  distinct  from  the  amboceptors  of  the 
hemolysins.  The  most  extensive  recent  work  on  the  hemopsonins  is  by  Hektoen.  ? 
He  shows  that  normal  serum  may  contain  hemopsonins  for  heterologous  or  even 
homologous  erythrocytes;  that  immune  hemopsonins  are  highly  resistant  to  heat;  and 
are  in  part  specific  and  in  part  non-specific. 

Methods. — The  body  fluids  of  the  dogs  were  secured  as  described  earlier  in  this 
paper,  and  inactivated  by  heat  at  55°  C.  for  30  minutes.  Rat  corpuscles  were  used 
throughout  the  work.  They  were  made  up  to  5  per  cent  suspension  after  careful 
washing  in  0.9  per  cent  NaCl  solution.  The  technic  followed  was  to  measure  into 
each  of  a  series  of  small  test  tubes  quantities  of  fluids  ranging  between  0.2  c.c.  and 
0.002  c.c.  and  then  adding  enough  salt  solution  to  make  0.2  c.c.  To  this  was  then 
added  0.4  c.c.  of  a  mixture  of  equal  quantities  of  rat  corpuscles  and  leukocytic  suspen- 
sion. In  this  way  dilutions  varying  between  1:3  and  1:300  were  secured.  The  tubes 
were  incubated  in  a  shaker  for  one  hour.  Then  the  contents  were  mixt  thoroughly, 
smears  made,  and  fixt  in  absolute  alcohol  for  one  hour,  and  then  stained  with  Giemsa's 
stain  for  30-60  minutes.  The  percentage  was  figured  from  the  number  of  the  leuko- 
cytes actively  phagocytic.  No  attempt  was  made  to  count  the  number  of  erythrocytes 
engulfed  per  leukocyte.  In  every  case  500  leukocytes  were  counted.  Our  figures  will 
represent,  then,  the  percentage  of  leukocytes  phagocytic,  and  shows  only  the  activity  of 
thermostable  hemopsonins. 

Early  in  the  work  an  experiment  was  performed  to  test  the  accuracy  of  the  method. 
Two. complete  sets  of  tests  were  made  with  the  fluids  of  a  normal  dog,  smears  were 


FRANK  C.  BECHT  AND  JAMES  R.  GREER 


made,  fixt,  and  stained  in  the  ordinary  way.  Labels  were  then  attached  to  the  slides 
and  one  of  us  numbered  the  slides  in  a  haphazard  manner  and  kept  a  record  of  the 
numbering  used.  The  slides  were  all  mixt  together  and  then  counted  by  the  other. 
In  this  way  the  subjective  factor  would  not  enter  at  all.  The  results  were  highly 
satisfactory,  for  by  counting  500  leukocytes  and  estimating  the  percentage  from  that 
number,  the  corresponding  slides  agreed  almost  exactly.  In  one  case  there  was  a 
difference  amounting  to  over  2  per  cent. 

NORMAL  DOGS. — The  first  point  was  to  determine  the  concentra- 
tion of  the  hemopsonins  in  the  body  fluids  of  normal  animals.  As 
a  typical  experiment  we  cite  Table  14. 

TABLE  14. 
COMPARATIVE  HEMOPSONIC  POWER  FOR  RAT  CORPUSCLES  OF  THE  BODY  FLUIDS  OF  A  NORMAL  DOG. 


Dilution 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericardial 
Fluid 

Cerebro- 
spinal  Fluid 

Aqueous 
Humor 

1:3... 

3  •  20 

i  .  20 

0.60 

o.oo 

0.40 

o.  20 

i-6 

O.2O 

o  oo 

o  20 

o  oo 

1:12  

0.20 

0.00 

0.00 

0.00 

0.00 

0.00 

Control =0. 

This  table  shows  that  the  concentration  of  hemopsonins  in  the 
various  body  fluids  runs  practically  parallel  with  that  of  the  other 
antibodies.  It  is  highest  in  the  serum.  The  two  lymphs  are  lower 
but  practically  equal.  There  are  also  traces  in  the  other  fluids  in  this 
case.  In  another  normal  animal  the  same  conditions  were  found. 

ACTIVELY  IMMUNIZED  DOGS. — In  establishing  immunity  for  the 
work  on  hemopsonins  we  followed  a  uniform  technic.  Each  animal 
received  intravenously  0.5  c.c.  of  a  5  per  cent  solution  of  washed  rat 
corpuscles  per  kilo  of  body  weight.  The  animal  was  operated  the 
loth  day  after  the  injection.  We  cite  the  experiment  in  Table  15 
as  typical. 

TABLE  15. 
COMPARATIVE  HEMOPSONIC  POWER  FOR  RAT  CORPUSCLES  OF  THE  BODY  FLUIDS  OF  AN  IMMUNE  DOG. 


Dilution 

Serum 
(Normal) 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericar- 
dial Fluid 

Cerebro- 
spinal 
Fluid 

Aqueous 
Humor 

•  3  
•6 

24.4 

45-o 
38  4 

43-4 
28  o 

39-4 
28  " 

25-  8 
15  o 

10.8 

2.6 

4.8 
i  .0 

:i2  

1.8 

24.6 

5  8 

5-0 
3  ° 

14.6 

2    2 

7.0 

2    O 

I  .  2 
O.  2 

0.0 
O.O 

:6o  

0.0 

2.6 

2.0 

o  8 

0.0 
O    O 

0.0 
O    O 

O.O 
O.O 

0.0 
O.O 

=  300  

0.2 

0.0 

0.0 

Table   15   shows  that  a  considerable  degree  of  immunity  was 
reached,   the  immune  serum  producing  a  much  higher  degree  of 


CONCENTRATION  OF  ANTIBODIES 


phagocytosis  than  the  normal.  The  concentration  was  highest  in 
the  serum;  the  two  lymphs  are  practically  parallel  with  a  slight  balance 
in  favor  of  the  neck  lymph,  because  it  not  only  produces  a  higher 
percentage  of  phagocytosis  in  the  lowest  dilution,  but  also  causes 
phagocytosis  in  a  much  higher  dilution.  The  pericardial  fluid  shows 
a  somewhat  lower  concentration  than  the  lymphs;  the  cerebrospinal 
fluid  and  the  aqueous  humor  contain  the  opsonin  in  lower  concentra- 
tion than  the  pericardial  fluid. 

In  six  experiments  with  immune  dogs  the  concentration  of  hemop- 
sonins  ran  the  course  shown  in  this  experiment.  We  find  some 
variations  in  the  cerebrospinal  fluid  and  the  aqueous  humor.  In 
three  out  of  five  cases  where  the  cerebrospinal  fluid  and  aqueous 
humor  were  tested  the  concentration  of  the  hemopsonins  was  higher 
in  the  former  than  in  the  latter.  The  fourth  case  is  the  one  cited  in 
Table  15,  and  in  the  fifth  case  neither  showed  hemopsonins.  In 
one  of  six  cases  the  hemopsonins  of  the  pericardial  fluid  and  aqueous 
humor  were  practically  equal  in  concentration. 

PASSIVELY  IMMUNE  DOGS. — Passive  immunity  was  produced  in 
the  same  way  described  under  bacterial  agglutinins.  We  cite  here 
as  an  example  the  results  obtained  in  the  case  of  a  dog  immunized 
by  injecting  intravenously  300  c.c.  of  the  blood  of  an  immune  dog 
after  first  removing  260  c.c.  by  bleeding. 

TABLE  16. 

COMPARATIVE  HEMOPSONIC  POWER  FOR  RAT  CORPUSCLES  OF  THE  BODY  FLUIDS  OF  A  DOG  PASSIVELY 

IMMUNIZED  TO  RAT  BLOOD. 


Dilution 

Serum 
(Normal) 

Serum 

Neck 
Lymph 

Thoracic 
Lymph 

Pericar- 
dial Fluid 

Cerebro- 
spinal 
Fluid 

Aqueous 
Humor 

•  3  •  •  • 

,5  A 

T  r    5 

,  f. 

_     Q 

:6                    

7    8 

:  12  

4  6 

!?    fi 

i  6 

2    6 

:24  

2    6 

i  6 

o  .0 

O.O 

:6o  

i  8 

o.o 

:  120  

o  6 

•300  

The  results  in  our  experiment  in  passive  immunity  show  first,  an 
increase  in  the  concentration  of  the  hemopsonins  in  the  serum,  and 
second,  a  marked  increase  in  the  concentration  of  the  antibodies  in 
the  thoracic  lymph,  bringing  it  up  to  a  concentration  equal  to  that  of 
the  serum,  while  the  concentration  of  the  hemopsonins  in  the  neck 


156  FRANK  C.  BECHT  AND  JAMES  R    GREER 

lymph  remains  low  and  from  a  comparison  with  other  animals  we 
would  say  that  the  injection  of  the  highly  immune  serum  had  had  no 
effect  upon  the  concentration  of  the  hemopsonins  in  the  neck  lymph. 
The  same  is  true  of  the  pericardial  fluid,  the  cerebrospinal  fluid,  and 
the  aqueous  humor,  where  the  concentration  is  no  higher  than  in  the 
normal  dog.  It  must  be  remembered  that  this  dog  showed  a  relatively 
high  hemopsonic  power  before  immunization  by  injection  of  the 
immune  blood.  We  have  other  experiments  which  show  the  same 
phenomenon. 

From  these  experiments  we  draw  the  following  conclusions  : 

1.  Hemopsonins  are  found  in  the  body  fluids  of  normal  dogs. 
The  concentration  is  highest  in  the  serum,  lower  in  the  thoracic  and 
neck  lymph,  which  run  almost  parallel,  and  are  found  in  the  other 
body  fluids  only  in  traces. 

2.  By  a  process  of  immunization  the  hemopsonins  of  the  body 
fluids  can  all  be  increased.     The  order  of  descending  concentration 
is  the  serum,  neck  lymph,  thoracic  lymph,  pericardial  fluid,  aqueous 
humor,  and  the  cerebrospinal  fluid.     Sometimes  the  arrangement  in 
the  scale  of  the  last  two  is  reversed. 

3.  Hemopsonins  can  pass  from  the  blood  into  the  thoracic  lymph, 
but  apparently  they  do  not  pass  into  the  lymph  of  the  head  region. 
The  concentration  of  the  hemopsonins  in  the  pericardial  fluid,  cere- 
brospinal fluid,  and  aqueous  humor  is  not  increased  in  24  hours  over 
the  normal  by  passive  immunization  as  in  our  experiments. 

GENERAL   CONCLUSIONS. 

In  the  normal  animal  the  concentration  of  hemolysins,  hemag- 
glutinins,  bacterial  agglutinins,  bacterial  opsonins,  and  hemopsonins 
decrease  in  the  body  fluids  in  the  following  order:  serum,  thoracic 
lymph,  neck  lymph.  Traces  of  hemagglutinins,  bacterial  agglutinins, 
and  opsonins  are  found  in  the  pericardial  fluid.  Traces  of  opsonins 
are  found  in  the  cerebrospinal  fluid  and  aqueous  humor.  No  pre- 
cipitins  for  rabbit  serum  were  found  in  any  of  the  body  fluids. 

Immunization  increases  the  concentration  of  the  antibodies  named 
above  in  the  fluids  in  which  they  are  found  in  the  normal  animal. 
Hemolysins  are  sometimes  found  in  the  pericardial  fluid,  and  in  a 
few  cases  traces  of  bacterial  agglutinins  in  the  cerebrospinal  fluid  and 


CONCENTRATION  OF  ANTIBODIES  157 

aqueous  humor.  Protein  precipitins  are  found  in  the  serum,  thoracic 
lymph,  and  neck  lymph,  the  concentration  being  lower  in  the  last  than 
in  the  first  two. 

The  immunity  is  specific  in  the  other  body  fluids  as  in  the  serum. 

The  introduction  of  an  immune  blood  into  a  normal  animal 
increases  the  concentration  of  the  bacterial  agglutinins  in  both  the 
lymphs  above  the  normal.  The  introduction  of  an  immune  blood 
does  not  increase  the  concentration  of  hemopsonins  in  the  neck  lymph, 
altho  the  concentration  in  the  thoracic  lymph  is  markedly  increased. 
The  concentration  of  bacterial  agglutinins  and  hemopsonins  in  the 
cerebrospinal  fluid  is  not  modified  by  passive  immunization. 

The  passage  of  antibodies  from  blood  to  the  lymph  is  a  relatively     A 
rapid  process. 

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